NON-INVASIVE METHOD FOR DIAGNOSING ALZHEIMER'S DISEASE IN A PATIENT

Abstract: The present invention provides non-invasive methods for diagnosing Alzheimer's disease in a living human subject. One method employs a non-invasive automated apparatus which can continuously monitor pupil diameter size over time; repetitively measure pupil diameter size over time for a pre-chosen duration ranging from about less than 1 second to about 5 minutes; and cumulatively record size information as it is obtained over time. A second method employs an apparatus which can repetitively measure pupil constriction velocity for a pre-chosen duration both before and after stimulation by visible light. Both methods require the administration of at least one neural transmitter mediator to a targeted eye of the living subject in an amount insufficient to cause marked changes in pupil diameter size over time in a person who is not afflicted with Alzheimer's disease. The marked hypersensitivity of an Alzheimer's dementia patient to the administered neural transmitter mediator serves as the means for identifying individuals afflicted with Alzheimer's disease.

Description: Non-Invasive Method for Diagnosing Alzheimer's Disease in a Patient Field of the Invention The present invention is concerned with non-invasive methods for the diagnosis of Alzheimer's Disease in a living human patient and is particularly directed to using changes in pupillary characteristics caused by the application of neural transmitter agonists or antagonists to Alzheimer's Disease patients as a means of diagnosis. Background of the Invention Alzheimer's Disease ("AD") is a dementing disorder characterized by progressive impairments in memory and cognition. It typically occurs in later life; and is associated with a multiplicity of structural, chemical and functional abnormalities involving brain regions concerned with cognition and memory. This form of dementia was first reported by Alois Alzheimer in 1907 when he described a disease of the cerebral cortex in a 51- year-old woman suffering from an inexorably progressive dementing disorder. Although other forms of dementia had been well characterized at the time of Alzheimer's clinical report, his patient was clinically and pathologically unusual, because of her relatively young age and the presence of the then newly described intra-cellular inclusions which have subsequently come to be known as neurofibrillary tangles ("NFTs"). In recognition of this unique combination of clinical and pathological features, the term "Alzheimer's Disease" subsequently came into common usage. Today, Alzheimer's Disease is considered to be one of the formcoming scourges of the 21st century. This is due in major part to the aging of the population in concert with data indicating a marked increase in the incidence of dementia with advancing age. Epidemiologic studies suggest that the dementia presently occurs in up to 10% of individuals over the age of 65 and it is estimated that in the United States alone, as many as 4 million individuals may be affected with Alzheimer's Disease. The cost of caring for such individuals is well over 80 billion dollars annually and increasing rapidly. Since the recognition of this form of dementia as a specific disorder, many different neurobiologic approaches have been undertaken to studying and understanding the nature and the mechanism of action for Alzheimer's Disease, with a view to possible future therapeutic treatments and/or prevention. Representative of me range and diversity of these many neurobiologic approaches are those listed within Table 1 below. Table 1 Neurobiologic Approaches to the Study of Alzheimer's Disease* Biologic Measures Methods Examples Brain metabolism In vivo imaging studies Reduced glucose utilization in neocortex, esp. parietal and temporal areas Histology of brain Histochemistry, /A4 immunoreactive plaques in immunocytochemistry neocortex and hippocampus Quantitation of pathology Morphometric methods Reduced number of neurons in basal forebrain cholinergic system Neuron size and shape Golgi Stains Abnormal dendritic arborizations Ultπistructure Electron microscopy, PHF in NFT and /A4 fibrils in immunocytochemistry plaques Transmitters and enzymes Assays of markers Reduced levels of ChAT, somatostatin, and CRF in cortex Receptors Binding Assays in vitro Reduced cortical somatostatin autoradiography receptors and increased cortical CRF receptors Proteins in abnormal Purification of constituents, Decoration of PHF with organelles analyses of proteins and other -mtineurofilament and antiau components, antibodies; tubulinlike immuirocytochemistry freeze- immunoreactivity in GVD; actin rracture/deep-eteh in Hirano bodies; /A4 in plaque cores and congo philic angiopathy Proteins and their Immunoblots, Phosphorylated 200-kD modifications immunocytochemistry, in neurofUament A68 and tau vitro incorporation of amino associated with NFT; aberrant acids processing APP and PrP amyloid Biologic Measures Methods Examples RNAs Hybridization on gels and in Reduced mRNA in some cells; situ; measurements of PrP and APP mRNA present in mRNAs and enzymes acting neurons on RNAs Genes Recombinant DNA Anonymous marker on technology chromosome 21 linked to familial AD; APP gene localized to chromosome 21 ABBREVIATIONS AD Alzheimer's disease /A4 -amyloid protein CHAT choline acetyltransferase CRF corticotropin-releasing factor GVD granulovacuolar degeneration kD kilodalton(s) mRNA messenger ribonucleic acid(s) NFT neurofibrillary tangle(s) PHF paired helical filament(s) PrP prion protein *Source: DEMENTIA (Peter J. Whitehouse, Ed.), F.A. Davis Co., Philadelphia, 1993, Chapter 3, pp. 56-57. In addition, a great many research studies and clinical investigations have been undertaken to study die structural deficiencies, chemical changes, and functional abnormalities both within the brain and within different populations of nerve cells. The depth of such investigations and studies are represented by the following publications: Dementia, (J. Whitehouse, Ed.), F.A. Davis Company, Philadelphia, 1993; Paykel, et al., Arch. Gen. Psychiat., 57:325-332 (1994); Amaducci, et al., Neurology, 56:922-931 (1986); McKhann, et al., Neurology 34:939-944 (1984), Heston et al.. Arch. Gen. Psychiatry 38: 1085-1090 (1981); Aging of the Brain (Gispen and Traber, editors), Elsevier Science Publishers, Amsterdam, 1983, pages 275-282; Heyman et al., Ann. Neurol 75:335- 341 (1984); Brayne C. and P. Calloway, Lancet 7: 1265-1267 (1988); Roth et al., Br. J. Psychiatry 749:698-709 (1986); Medical Research Council, Report from the NRC Alzheimer's Disease Workshop, London, England, 1987; Morris et al., Neurology 47:469- 478 (1991); Alzheimer's Disease: Senile Dimentia and Related Disorders (Katzman, T.D. and R. L. Bick, editors), Raven Press, New York, 1994, pages 47-51; and the references cited within each of these publications. In spite of the many research investigations and diverse studies undertaken to date, present clinical evaluations still cannot establish an unequivocal diagnosis of Alzheimer's Disease. To the contrary, the only presently known means for positively proving and demonstrating Alzheimer's Disease in a patient can only be achieved by a brain biopsy or a postmortem examination to assess and determine the presence of neurofibrillary tangles (NFT) and senile (amyloid) plaques in brain tissue. These criteria for the definite diagnosis of Alzheimer's Disease are met only by histologic evidence. Instead, a set of criteria for the diagnosis of probable Alzheimer's Disease have been described and include: (1) the presence of a dementia syndrome with defects in two or more areas of cognition; (2) progressive worsening of memory and other cognitive function over time; (3) a relatively intact level of consciousness; (4) age at disease onset at a time between 40 and 90 years of age; and (5) the specific absence of any other systemic or central nervous system process that could account for the progressive cognitive deterioration in the individual. In addition, the probability of an accurate diagnosis in the living patient is augmented by laboratory examinations (such as VDRL and TFT) and by imaging studies (such as computed tomography and magnetic resonance imaging). Such laboratory examinations and/or imaging studies demonstrate the existence and effects of other causes of dementia (such as subdural hematoma, intracranial tumors, infection and brain infarction) and disclose results which are consistent with but are not themselves diagnostic of Alzheimer's Disease. Nevertheless, present clinical diagnoses are wrong in as many as 55 % of cases. Thus, mere is no sound basis or reliable test methodology at all today for the diagnosis of definite Alzheimer's Disease other than the microscopic examination of histologic evidence from brain biopsy or autopsy material. Instead, the best clinical diagnosis available to date is only a presumptive determination based on criteria which are evaluations of cognitive and neurological functions for that patient. It is therefore overwhelmingly clear that there has been and remains today a long standing need for an accurate method to diagnose Alzheimer's Disease clinically in a living human subject with substantial certainty and reliability. In addition, were such a diagnostic methodology also able to be non-invasive, rapid in time required for performance, and precise via the accumulation of large quantities of empirical data, such a diagnostic methodology would be recognized by physicians and laymen alike as being a major advance and substantial improvement in this field. The mydriatic response to eyedrops of the anticholinergic agent tropicamide at very low concentration (0.01 %) has been studied in patients with Down's syndrome, with a report that, in comparison with normal subjects, Down's syndrome patients had responses three times greater than normal patients. Sacks, et al., J. Neurology, Neurosurgery, and Psychiatry, 1989:52, 1294-1295. Sacks, et al. state that since patients with Alzheimer's disease appear to show behavioral and cognitive sensitivity to hyoscine, the eyedrop test could be used to distinguish people with Alzheimer's disease from patients with other forms of dementia, allowing for a definitive diagnosis in a living patient. U.S. patent 5,297,562 states that Alzheimer's disease can be diagnosed before symptoms of dementia occur by determining whether a patient is mosaic for trisomy 21, including by measuring certain characteristics known to be associated with Down's syndrome. Among the characteristics listed by Potter are pupil dilation, with citation to Sacks, et al. United States Patent Application Number 08/109,746, filed August 20, 1993, apparently relates to the use of a mydriadic response to diagnose Alzheimer's disease in a patient, including methodologies that use an untreated eye as a control for deteπnining die mydriatic response in a treated eye. Summary of the Invention The present invention provides non-invasive methods for diagnosing Alzheimer's Disease in a patient, i.e., living human subject. In particular the present invention provides: A non-invasive method for diagnosing the presence or absence of Alzheimer's disease in a living subject which comprises: administering to one of said subject's eyes at least one neural transmitter mediator in an amount insufficient to cause a significant pupil constriction or dilation if said subject is not afflicted with Alzheimer's disease, repetitively and episodically measuring pupil diameter in said treated eye during at least a part of the time said neural transmitter mediator or mediators would have an observable effect on pupil diameter in a subject afflicted with Alzheimer's disease, and diagnosing the presence or absence of Alzheimer's disease in said subject based on the presence or absence of Alzheimer-characteristic pupil diameter changes from a baseline pupil diameter established for said treated eye or Alzheimer 's-characteristic pupil diameter rates of change calculated from said pupil diameter change measurements. In addition to the above diagnostic method, the present invention further relates to a method, as described above, that additionally comprises: photostimulating said subject's treated eye with one or more episodes of visible light to induce pupillary constriction, such that said episodic measurements of pupil diameter occur during the pupil contriction in response to said photostimulation, said pupil diameter measurements being thereafter used to determine the rate of change of pupil constriction (pupil constriction velocity). In its most useful embodiment, the present invention is practiced in a manner in which said repetitive measurments occur at a high frequency, i.e, at least 50 Hertz, over a duration sufficient to provide a statistically meaningful measure of the pupil diameter dynamic used as the diagnostic determinant. Ordinarily, therefore, the diagnostic method of this invention will assess actual pupil diameter from at least 1000 separate measurements of pupil diameter, or at least 100 separate measurements each time photostimulated constriction velocity is measured. Preferably, each of these high-frequency measurements is repeated multiple times, extending at least up to the time of maximum expected sensititivity of the subject to the mediator. Thus, ordinarily, three to six post- administration episodes of high frequency measurement would be undertaken, in addition to one or more baseline measurements. As noted above, the changes in pupil diameter (e.g., maximal change in pupil diameter using the post-administration high-frequency measures compared to the high- frequency baseline measure) or photostimulated pupil contriction velocity are then matched with Alzheimer's-characteristic measures for these parameters to assess the likelihood that the subject is afflicted with Alzheimer's disease. These Alzheimer's-characteristic values are readily and empirically determined since Alzheimer's patients typically exhibit highly statistically significant different responses when the high-frequency, multiple-episode SUBSTITUTE SHEET (RULE 261 measures are undertaken. For example, the separation of dynamic values as between normal patients and Alzheimer's-afflicted patients is normally at least 10% and typically at least 15% or greater following administration of 0.01 % tropicamide, allowing for a direct statistical correlation between the pupil dyanmic change and the likelihood (probability) that subject has Alzheimer's disease. In its actual operation, the invention includes a diagnostic method comprising the steps of: providing a non-invasive automated apparatus which can continuously monitor pupil diameter size over time, repetitively measure pupil diameter size over time for a prechosen duration ranging from about less than 1 second to about 5 minutes, and cumulatively record such monitored and measured pupil diameter size information as is obtained over time; identifying one eye in the living human subject as a targeted eye; using said non-invasive automated apparatus on a first measurement occasion to continuously monitor, repetitively measure, and cumulatively record the pupil diameter size of said targeted eye in the living human subject over the prechosen duration to provide primary informational data of pupil diameter size for said targeted eye; administering at least one neural transmitter mediator to said targeted eye of the living human subject in an amount insufficient to cause marked changes in pupil diameter over time in a person not afflicted with Alzheimer's disease, said neural transmitter mediator being selected from the group consisting of cholinergic and adrenergic antagonists and agonists; waiting a predetermined interval of time sufficient for said administered neural transmitter mediator to act upon said targeted eye; then using non-invasive automated apparatus on at least a second measurement occasion to continuously monitor, repetitively measure, and cumulatively record the pupil diameter size of said targeted eye in the living human subject over the prechosen duration to provide at least secondary informational data of pupil diameter size for said targeted eye after being acted upon by said administered neural transmitter mediator; and determining at least one parameter selected from me group consisting of pupil diameter dilation, pupil diameter constriction, and me rate of pupil diameter change for said targeted eye as occurred over said time interval by comparing said primary informational data with at least said secondary informational data, whereby a marked increase in pupil diameter size, or a marked decrease in pupil diameter size, or rapid rate of pupil diameter change for said targeted eye diagnostically establishes the living human subject as being afflicted with Alzheimer's disease. Alternatively, the invention can be practiced using photostimulation to measure pupil constriction velocity in the following manner: providing non-invasive apparatus means for (a) introducing photostimulating visible light of predetermined wavelength and intensity to the eye on-demand sufficient to cause a constriction of the pupil, and (b) determining the velocity of pupil constriction caused by said introduced photostimulating visible light; identifying one eye in the living human subject as a targeted eye; administering at least one neural transmitter mediator to said targeted eye of the living human subject in an amount insufficient to cause a marked change in pupillary dynamic response in a person not afflicted with Alzheimer's disease, said neural transmitter mediator being selected from the group consisting of cholinergic antagonists and agonists; waiting a predetermined interval of time for said administered neural transmitter mediator to act upon said targeted eye; then introducing photostimulating visible light of predetermined wavelength and intensity to the targeted eye sufficient to cause a constriction of the pupil using said non-invasive apparatus means; and determining pupil constriction velocity for said photostimulated targeted eye using said non-invasive apparatus means, a marked decrease in pupil constriction velocity for said targeted eye with respect to a pre-established normative standard diagnostically establishing that living human subject as being afflicted with Alzheimer's disease. Brief Description of the Figures The present invention can be more easily and completely understood when taken in conjunction with the accompanying in which: Fig. 1 is a schematic illustration of the series 4000 measurement apparatus as a system. Fig. 2 is a schematic illustration of the optics and the series 4000 measurement apparatus as an integrated system. Fig. 3 is a graph illustrating the differences in pupil dilation responses to a cholinergic antagonist between patients with clinically determined Alzheimer's disease and cognitively intact elderly individuals obtained using the present methodology. Results for patients clinically diagnosed as having Alzheimer's disease are denoted by darkened circles and results for cognitively intact elderly individuals by open circles. Fig. 4 is a graph illustrating the differences in pupil dilation response to a cholinergic antagonist among patients with clinically determined Alzheimer's disease, suspect Alzheimer's individuals, cognitively abnormal elderly subjects, patients with non- Alzheimer's dementia, and cognitively intact normal persons obtained using the present methodology. Symbols are as follows: darkened circles - results for patients clinically diagnosed as having Alzheimer's disease; darkened triangles - results for subjects suspected of having Alzheimer's disease; darkened squares - results for cognitively abnormal elderly persons; open circles - results for cognitively intact elderly persons; open squares - results for patients with non-Alzheimer's dementia. Fig. 5 is a scatter plot display illustrating pupil response data for the 29 minute sampling occasion for clinically determined Alzheimer's patients and cognitively intact elderly control subjects. Abbreviations are as follows: NC= normal control subjects; AD/SAD = Alzheimer's disease patients/suspect Alzheimer's disease individuals; CAE = cognitively abnormal elderly persons; NAD = non- Alzheimer type dementia patients.. Fig. 6 is a graph illustrating the mean scores and +/-95% confidence limits of patients with probable Alzheimer's disease and cognitively intact elderly individuals obtained using the present methodology. Darkened circles represent results for patients with probable Alzheimer's disease and open circles represent represent results for cognitively intact elderly persons. Definitions In order to avoid ambiguity in terminology as well as to provide a clear and precise understanding of the scope of me present methodology, a set of specific terms and explicit definitions are given below. These words and their meanings will be employed repeatedly and routinely in this disclosure and the stated definitions are to be accepted as written and as part of the general lexicon and vocabulary in this art. Cholinergic: A term pertaining to me neural transmitter acetylcholine. The term is particularly used to designate nerve fibers diat release acetylcholine at u eir terminals, or the physiological effects produced by the stimulation of these nerve fibers, or me acetylcholine receptors on the post synaptic membrane, or chemical agents and drugs that imitate the effects of acetylcholine. Adrenergic: A term designating activation by, characteristic of, or a secreting of the neural transmitter epinephrine or substances with similar epinephrine-like activity. The term is also used to refer to those postganglionic sympathetic nerve fibers that liberate norepinephrine in response to a nerve impulse; and is typically used to identify an agent that produces such an effect. Mydriasis: Dilation of the pupil. Mydriatic agent: A compound or substance which initiates, induces, promotes or causes pupil dilation. Miosis: Constriction of the pupil. Miotic agent: A compound or substance which initiates, induces, promotes, or causes pupil constriction. Neural Transmitter (also Neurotransmitter and Synaptic Transmitter): A compound or substance that serves to transmit a nerve impulse between cells at a synapse or a neuromuscular junction. Such compounds include but are not limited to acetylcholine, epinephrine, norepinephrine, dopamine, serotonin, γ-aminobutyric acid, glycine, and glutamate. Constriction Velocity: The average rate of change in pupil diameter expressed in mm/sec over a given interval of time from initial size to maximal constricted size of pupil. Re-dilation velocity: The rate of recovery expressed as mm/sec to maximal resting pupil diameter after stimulation by light. Endogenous substance: A compound or composition developing or originating within the person or arising from causes within the person's body. Exogenous substance: A compound or composition synthesized, found, or originating outside the person's body. Photostimulation (visible light stimulation): the purposeful introduction of visible light energy to the eye of a living subject. Ligf energy (photoenergy): Electromagnetic radiation of any wavelength including infrared, visible and ultraviolet wavelengths. Agonist: A compound or substance that imitates, mimics, or acts in a manner similar to the activity or function of a specified tissue, composition or agent. Antagonist: A compound or substance that blocks the activity or function of a specified tissue, composition or agent. Mediator: A compound, composition, agent or substance that influences, effects, intervenes, contradicts, mitigates, modifies, promotes, or is involved wim an activity or function in a specified manner. Velocity: The rate of change of size or rate of displacement typically expressed in unites (e.g., millimeters per second). Velocity is a vector quantity and a complete specification which states both t e direction as well as the magnitude of change. Detailed Description of the Invention The present invention is directed to non-invasive methods for diagnosing Alzheimer's disease in a living human subject. As will be described in detail below, these diagnostic methods take advantage of a new recognition that some aspects of the typical Alzheimer Disease patient's autonomic nervous system are hypersensitive to neural transmitter mediators administered specifically to the eye. In one method, measurements are made of changes in pupil diameter in response to neural transmitter mediators (cholinergic or adrenergic agonists and antagonists) when such mediators are applied at concentrations known to be too low to significantly affect pupil diameter in normal individuals. In an alternative method, precise measurements are made of pupil constriction velocity in response to stimulation by a predetermined intensity and duration of visible light energy. The method utilizes differences in pupillary dynamic response to stimulation by a known quantity of light between Alzheimer's patients and cognitively intact persons to such pharmacologically active agents as an empirical basis and standard for accurately, positively and definitely diagnosing Alzheimer's dementia. When performed in accordance with the prescribed manipulative steps, the present diagnostic methods provide many major benefits and multiple advantages which were not known or available previously. These include the following: 1. The present diagnostic methods are relatively non-invasive. The methodology utilizes automated equipment which can repetitively measure pupil diameter size changes over time and cumulatively record such data as it is obtained or which can accurately monitor pupil constriction velocity in response to light stimulation. The data so obtained can then be mathematically analyzed to provide a quantitative clinical result for comparison with an established numerical standard range of normal and abnormal values. In this manner, a definite, unambiguous, and reliable determination can be made as to whether or not that living human subject is afflicted with Alzheimer's Disease. 2. The use of automated equiptment to monitor and record changes in pupil diameter size and pupillary responses provides a large quantum of empirical data which can be used for making a clinical diagnosis. The automated equiptment is able to observe and measure pupil diameter size and constriction velocity quickly, accurately, and repetitively. For example, one of the devices described herein will provide 50 to 60 measurements per second for any desired duration of time. If the duration of measurement were extended for about 30 seconds, this would yield as many as 1,800 individual determinations. While he duration of pupil diameter measurements may vary considerably, there will be in each instance a quantity of raw data which far exceeds that reasonably obtainable using non- automated methods. This improves the accuracy and reliability of measurements. 3. The diagnostic methodology as a whole makes minimal demands upon the patient; does not involve physical exercise or fatiguing mampulations; and avoids the use of systemic medication. Instead, die present methods rely upon the use of dilute concentrations of neural transmitter mediators such as cholinergic antagonists and agonists and adrenergic antagonists and agonists. These neural transmitter mediators are adπiinistered to test subjects at concentrations which do not substantially influence pupillary responses in cognitively intact individuals. Accordingly, there is little or no probability that die diagnostic examination process 'will cause undesirable side effects. 4. The present diagnostic methods may be performed relatively easily and can be completed within about an hour. The diagnostic data are then generated as quickly as the central processing unit of the automated apparatus can operate; and the results appear in printed form or in visual form on a monitor and/or may be transferred to a remote reference facility for final analysis as is most desired or required under the use circumstances. 5. The diagnostic memods employ a pharmacological manipulation of the pupil of the eye. In one preferred method, a neurotransmitter mediator (cholinergic or adrenergic antagonist or agonist) is applied to one targeted eye of an individual being tested and his pupillary response (percent change over baseline or rate of change) is compared to norms established for a population of, preferably, age-matched, cognitively intact individuals. A significant difference from the established norm in the pupillary response of such an individual serves to diagnose Alzheimer's disease. In a second preferred method, a cholinergic antagonist is applied to a subject's eye and the constriction velocity of his pupil in response to light stimulation is compared to the constriction velocity of normal individuals. Again, it is preferred that subjects be compared to an age matched normal population. A significant difference in constriction velocity would be indicative of Alzheimer's disease. An alternative and less desirable method uses one eye of the individual as the targeted eye for treatment with neural mediators while the other eye is employed as an untreated control. In this manner, the difference in pupillary response is thus measured between the two eyes of the same living subject. 6. The present diagnostic methodology can be performed and practiced in several different modes. These procedures include measuring pupil diameter dilation; pupil diameter constriction; die rate of pupil diameter changes; and photostimulation-induced pupil velocity contriction. The scientific basis for the present diagnostic methods is the recognition that persons afflicted with Alzheimer's dementia are uniquely hypersensitive to the pharmacological effects of neural transmitter mediators, particularly those administered topically to the eye. The existence of such hypersensitivity, however, is not only an intrinsic part of die disease process but also can be intentionally manipulated pharmacologically in the person afflicted with Alzheimer's Disease. Thus, the underlying principles for the present invention are first, that this unique hypersensitive state exists and manifests itself in the autonomic nervous system of the Alzheimer's patient; and second, that this hypersensitive state will manifest itself as an abnormal response to pharmacologically active antagonists and agonists in a distinctive manner which can be utilized for diagnostic purposes. The present disclosure provides, for the first time, an empirical showing that hypersensitivity to neural transmitter mediators is specifically present in those neurons and nerve cell bodies which innervate the iris muscle of the eye. Thus, me administration of a neural transmitter mediator or modular (such as cholinergic or adrenergic antagonists or agonists) in a concentration which is generally insufficient to cause a marked or noticeable dilation or contraction of pupil diameter size in a cognitively intact individual -will nevertheless cause a change and marked alteration in pupil diameter size (dilation or contraction) in die person afflicted witii Alzheimer's Disease. Thus, the underlying principle of the present methods is that an intrinsic part of the Alzheimer's disease process is hypersensitivity to neural transmitters affecting the pupil; and the concomitant demonstration that pharmacologically active mediators can be employed to manipulate this disease condition and to yield a hypersensitive pupillary response. The present invention proves that these circumstances exist within the Alzheimer's population generally and thus may properly serve as a basis for making a differential diagnosis and determination by which to identify the presence of Alzheimer's Disease in a single living human individual. In order to more fully understand and appreciate the present invention, the detailed disclosure below is divided into two sections. Section I describes die diagnosis of Alzheimer's Disease based upon neurotransmitter-stimulated changes in pupil diameter. Section II describes the diagnosis of Alzheimer's disease based upon light-stimulated pupil constriction velocity. Section I: Diagnosis of Alzheimer's Disease Based Upon Neurotransmitter-Stimulated Changes in Pupil Diameter A. Modes for Performing the Diagnostic Method The present diagnostic methodology can be performed in any of at least three different modes; and within each of these alternative modes, at least two major variants are available regarding me class of neural transmitter mediator used and me manner in which changes in pupil diameter are observed and determined. Each of the three different alternative modes and the individual categories or classes of neural transmitter mediator are described in detail below. 1. Mode 1 : Pupil Dilation (Mydriasis) Apart from the memodology described in section II below, the most preferred mode of performing the diagnostic memodology utilizes the percentage change in pupil dilation as me diagnostic feature of choice. Pupil dilation is me response most easily observed and measured; and pupil dilation provides me greatest possible range of pupil diameter size changes and variances for the population as a whole. In order to permit pupil dilation (ratfier than any other type of pupillary change) to occur at all as a demonstration of Alzheimer's dementia hypersensitivity, one must administer a dilute concentration of at least one neural transmitter mediator which is a recognized and conventionally known mydriatic agent. Typically tiiis will be an agent selected from the group consisting of cholinergic antagonists, adrenergic agonists, or a combination of these agents. A representative, but non-exhaustive listing of these drugs is provided in Table 2 below. Table 2: Neural Transmitter Mediators A. Anticholinergic Agents Name Brand Conventionally Present Use/ Agent-Generic Example Used Doses Comments Tropicamide Mydriacyl 0.5-1.0% Usually 1.0% Atropine Atropisol 1 % Not routinely used for eye examinations in adults. Homotropine I-Homatrine 2% q 10-15 min Used for refraction Hydrobromide not dilation Cyclopentolate Cyclogyl 0.5-2% 0.5% for Hydrochloride fundoscopic examination Scopolamine Isopto Hyoscine 0.2-0.25% Used for post-op mydriasis not eye examinations B. Adrenergic Agents Name Brand Conventionally Present Use/ Agent-Generic Example Used Doses Comments Phenylephrine Mydfrin 2.5% — Hydrochloride Hydroxyamphe- Paredrine 1 % amine Hydrobromide Dipivefrin Propine 0.1 For the treatment of glaucoma Epinephrine Epifrin 1-2% up to 4x/day Comhined Agents Name Brand Conventionally Present Use/ Agent-Generic Example Used Doses Comments Cyclopentolate Cyclomydril 0.2% cyclopen & Hydrochloride & 1/% phenyleph. Phenylephrine 2. Mode 2: Pupil Contraction (Miosis) An alternative mode for performing the diagnostic method utilizes pupil constriction as die diagnostic feature which is measured. Pupillary constriction is a second form of hypersensitive reaction demonstrated by an Alzheimer's disease patient as a consequence of the administration of another class of neural transmitter mediator-the miotic agent. In order to allow pupillary constriction to occur as a manifestation of the hypersensitivity generated by Alzheimer's disease, one must administer a dilute concentration of at least one miotic agent to the eye of the subject under test. The pharmacologically active compounds are cholinergic agonists or adrenergic antagonists; and typically include die classes of parasympathometic agents, short-acting anticholinesterase agents and long-acting anticholinesterase agents. A representative, but non-exhaustive, listing is provided by Table 3 below. Table 3: Miotics Parasvmpathomimetic Agents Name Brand Conventionally Present Use/ Agent-Generic Example Used Doses Comments Pilocarpine Pilocar 14% solution For die treatment Hydochloride 1-6 times/day of glaucoma Pilocarpine Pilagan Nitrate Liquifilm Carbachol Isopto 0.75-3% solution For the treatment Carbachol 3-6 times/day of glaucoma B. Short- Acting Anticholinesterase Agents Name Brand Conventionally Present Use/ Agent-Generic Example Used Doses Comments Physostigmine Eserine Sulfate 0.25% solution For the treatment Sulfate/ 4+ times/day of glaucoma Physostigmine Isopto Eserine Salicylate Long-Acting Anticholinesterase Agents Name Brand Conventionally Present Use/ Agent-Generic Example Used Doses Comments Demecarium Humorsol 0.125% =0.25% For the treatment Bromide q 12-48 hours of glaucoma Echothiophate Phospholine 0.03%-0.6% For the treatment Iodide Iodide q 12-48 hours of glaucoma D. Beta-Adrenergic Antaponists Name Brand Conventionally Present Use/ Agent- Generic Example Used Doses Comments Timolol Maleate Timoptic 0.25%-0.5% For the treatment 2 times/day of glaucoma 3. Mode 3: Rate of Pupil Diameter Size Changes A third mode of performing the diagnostic method of the present invention utilizes he rate of change in pupil diameter size as the diagnostic feature. A markedly rapid velocity in pupil diameter change after administration of a neural transmitter mediator serves to identify the hypersensitive reaction of the person afflicted with Alzheimer's disease and distinguishes that person's pupillary response from those of cogmtively intact persons. Clearly, the rate of change is the critical factor; and the pupil can be either dilated or constricted during the performance of the procedure in order to make the determination. The mode of observation and measurement is the direct comparison of the rate of change for an individual being tested for Alzheimer's disease to those rates of change established in a population of age-matched and cognitively intact individuals. A substantive difference in rate of change from the population normal range of values would serve to diagnose Alzheimer's disease in the tested individual. A less desirable but alternative basis of evaluation would compare the rate of pupillary change for the treated eye against die rate of change for the untreated eye in die same individual. C. Use Parameters And General Guidelines For Practicing The Diagnostic Method A range of general guidelines and use parameters are provided herein for the optimization and convenience of bo d e user and the individual being tested. These general guidelines are provided for the benefit of the intended user and are merely illustrative examples to consider when preparing detailed protocols intended for use on a clinical basis. 1. Duration Of The Sampling Occasion. An essential part of the present methodology is the use of a non-invasive automated apparatus to continuously monitor and repetitively measure pupil diameter size over time for a prechosen duration ranging from less than 1 second to about 5 minutes (300 seconds). Each continuous observation and repeated determination of pupil diameter size over time constitutes one "sampling occasion." As is described in greater detail hereinafter, the preferred automated apparatus is able to monitor and measure pupil diameter size repeatedly and continuously at a rate of at least 60 determinations per second. However, automated apparatuses which perform die requisite functions at a slower rate (e.g., less man 20 determinations per second) can also be used. It will be readily recognized that the quantum of data available for analysis will increase as the duration of measurement increases. Thus, for data obtained at a rate of 60 determinations per second, a one second duration yields 60 individual measurements of pupil diameter size whereas the preferred duration of about 30 seconds would yield 1800 individual measurements of pupil diameter size, with the entire 30 second interval constimting one sampling occasion. Thus, longer duration times produces more data for subsequent mathematical analysis. The present invention requires that sufficient data be obtained to provide a statistically meaningful measure of pupil diameter. Low frequency measures or short durations of measurement provide insufficient data for statistical purposes and, ordinarily, will fail to provide a meaningful or reliable diagnosis. Accordingly, as noted above e present invention provides for repetitive measurements during each sample occasion or episode. Using a convenient sampling frequency of 60 Hertz allows for a reasonable permits statistically useful data to be obtained provided that sampling is continued for preferable at least 20 and more preferably at least 30 seconds. The present methodology allows for a choice in he duration of time constituting one "sampling occasion. " This duration of time should be kept reasonably constant and uniform during the entire diagnostic protocol and will constitute die standard number of seconds or minutes for each sampling occasion. In addition, die full chosen duration of time constimting the sampling occasion may be achieved in two, alternative, formats: as a single, uninterrupted interval time for continuous monitoring and repetitive measurement; or as a series of discrete time aliquots in sequence with slight pauses (typically seconds) interrupting the monitoring and measurement process. With d e illustrative preferred automated apparatus providing 60 determinations per second, a 30 second duration sampling occasion is deemed to be adequate and reliable for diagnostic determination purposes. Alternatively, if the apparatus at hand is a much slower operating instrument offering-for example, only 20 determinations per second- then a somewhat longer duration of time constituting each sampling occasion interval may be desirable. Similarly, witii me advent and manufacture of ever more rapid automated instrumentation, measurement speeds far greater tiian 60 determinations per second are envisioned, and accurate determinations may be made at sampling occasions of shorter duration. 2. Frequency Of Sampling Occasions At least two sampling occasions, separated by a prechosen length of time, should be made when practicing the present diagnostic method. The first sampling occasion constitutes the "zero" time and provides the initial baseline characteristics of untreated pupil diameter size for that individual patient. It is expected and intended that this initial baseline sampling occasion be made on bo the left eye and d e right eye of the patient. In general, either of die two eyes may be used as die targeted eye for subsequent treatment with a neural transmitter mediator. The untreated control eye in turn will receive a drop of sterile water. After performing die baseline measurement, the present diagnostic metiiod requires that at least a second sampling occasion be performed, preferably when the maximum difference in pupil diameter change or pupillary response occurs. The methodology therefore requires at least two different sampling occasions of specified duration during which the treated targeted eye and desirably the non-treated controlled eye are monitored and measured. In preferred protocols, however, from 3 to about 6 different sampling occasions are performed over a period of about one hour. The greater number of sampling occasions within the overall protocol will allow a plotting and empirical determination which more accurately identifies the pupillary response peak or die maximal effect of treating die targeted eye with die chosen neural transmitter mediator. Thus, d e preferred protocol will have 6 different sampling occasions of, for example, 30 seconds duration each (assuming the automated apparatus can provide a capability of 60 determinations per second). 3. Concentration of die Chosen Neural Transmitter Mediator In the preferred mode of practicing die present diagnostic methodology, die concentration of neural transmitter mediator should be such that it will not markedly affect the pupillary responses of cognitively intact people, not afflicted with Alzheimer's dementia. For example, it has been empirically determined tiiat the concentrations of tropicamide which are insufficient to cause such pupillary responses in normal individuals includes a range of concentration from 0.0025% to about 0.01%. Thus, the range of tropicamide concentrations may be used in die present method includes 0.0025 % , 0.005 % , and 0.01% . Depending on the concentration of neural transmitter mediator actually used, the amount of time before pupillary responses show die most significant differences between Alzheimer's patients and normal controls may differ; and thus the time of maximal response may vary. For example, it is estimated tiiat a concentration of 0.01 % tropicamide will result in a maximum pupillary dilation response at about 29 minutes after its administration to die targeted eye in an Alzheimer's patient. Thus, the repetitive and episodic measurements of pupil diameter would continue up to and including this period of expected maximal response to facilitate determination of d e most significant possible difference between a possible Alzheimer's patient and the established value for a normal control. It will also be appreciated that in the less desirable modes, die test method can employ a use concentration or strength of neural transmitter mediator which is sufficient to cause some noticeable change in pupil diameter and pupillary response even in a normal, non- Alzheimer's individual. 4. Preferred Basis For Comparing The Empirically Obtained Result In Order To Diagnose Alzheimer's Disease A diagnosis is preferably made when the change (or rate of change) in pupil diameter of die eye treated with eitiier a cholinergic or adrenergic antagonist or agonist exceeds a predetermined range of numerical values representative of die cogmtively intact population as a whole. This range of numerical values is considered die diagnostic criterion for determining die presence or absence of Alzheimer's disease in a living human individual. The diagnostic evaluation is empirically determined by examining die percentage change in pupil diameter (or rate of change) for diagnosed Alzheimer's patients and for known cognitively intact individuals for a particular neural transmitter substance at a particular concentration; and determining me point at which known Alzheimer's patients compared to known cognitively intact individuals are distinguishable. D. Preferred Protocols It will be appreciated that the preferred protocols presented herein are merely illustrative of die diagnostic methodology as a whole and are intended to be modified in a substantive manner to practice the different modes described previously herein as well as to accommodate different automated equipment and clinical circumstances. The preferred protocols employ the pupil dilation mode of analysis; utilize dilute concentrations of a cholinergic antagomst as the pharmacologically active neural transmitter mediator; and use either a series 4000 TV based eye measurement system or PUPILSCREEN pupillometer as me automated apparatus for taking repeated diameter size measurements. 1. A First Preferred Protocol for Performing the Diagnostic Methodology a) Prior to beginning me pupil assay, the following patient screening tests must be done. l. Evaluate d e patient for any ocular abnormalities: a. cataracts. b. history of glaucoma. c. a narrow anterior chamber. 2. If patient exhibits condition "c" do not proceed with the test. 3. If patient has cataracts that distort die shape of die pupil excessively do not administer die neural transmitter mediator to die affected eye. 4. Screen patients for any current use of drugs whh central or peripheral cholinergic effects. If patient is currently using medications with known cholinergic effects note on patient record for future reference in interpreting pupil assay data. b) Once screening has been done, insure that the patient is alert and not agitated or drowsy. If patient is excessively drowsy do not proceed wid test, but schedule die patient for future testing. c) Allow three minutes for die patient to sit quietiy while pupils adjust to ambient photopic illumination of no greater than 5 foot candles in die examining room. d) After three minutes, image the patient's eye widi a series 4000 TV based eye measurement system. Set die pupil discriminator so that die eye is completely encircled widi the white discriminator and forms a clean elliptical image in die center of the pupil monitor. Open a data file and begin recording pupil diameter. Close die data file after measurements have been recorded for 1 minute. Open a second file and image the other eye as described above. Record 1 minute of baseline data. Each minute of observation will yield 3600 individual measurements of pupil diameter. e) After completing the baseline readings and saving this data to disk, administer a single drop of die chosen neural transmitter mediator at die appropriate use concentration (e.g.. a .01 % tropicamide solution) to one targeted eye, chosen arbitrarily. The drop should be administered in the following manner: 1. Have the patient in a position on a chair or an examining table so that they can tilt their head well back. 2. Hold open d e lower and upper eyelid with die thumb and first finger. 3. Squeeze the bottle of treatment solution gently so as to allow a single drop to fall on d e center of die lens of the eye. 4. Have die patient close his/her eye after administration of the drop. 5. Administer gende pressure on d e inner canthus of the eye for 1 minute to prevent excessive entry into die systemic circulation. f) After 1 minute have die patient sit up. Wait one minute for the eye to adjust to ambient illumination and proceed to image die pupil as described in d) above. Record 30 seconds of pupil diameter to a data file. This duration of pupil diameter observation and measurement will yield approximately 1800 individual determinations of size. g) Repeat the procedure in steps e) and f) widi die other non-treated eye but using a single drop of sterile water for ophthalmic use in the place of neural transmitter mediator. h) After administration of die sterile water drop to the non-treated eye and measurement of die non-treated pupil, have die patient wait quietly for a period of five minutes while viewing a video tape on he CRT monitor, i) After 5 minutes have elapsed, turn off die video tape and wait for 1 minute while die patient's eye accommodates to the low illumination. Open a data file and image the eye as described in d). Record pupil diameter from die treated eye for 30 seconds. Repeat this procedure widi die untreated eye. Each 30 second pupil diameter measurement occasion will yield approximately 1800 individual readings of pupil diameter size for each eye. j) Repeat diis procedure every 5 minutes until minute 30. k) After die last reading at minute 30 have the patient view the video tape for 10 minutes. After 10 minutes record pupil diameter from the treated and untreated eyes as described above. 1) Have d e patient view a final 10 minute segment of video tape and record pupil diameter from the treated and untreated eyes as described above. The final reading should be taken approximately 55 minutes after administration of die eye drops. 2. A Second, Alternative Preferred Protocol for Performing me Diagnostic Methodology a) Screen patients as described above in step a) of the first preferred protocol. b) Once screening has been done insure that the patient is alert and not agitated or drowsy. If patient is excessively drowsy do not proceed widi test, but reschedule die patient for future testing. c) Allow three minutes for patient to sit quiedy while pupils adjust to ambient photopic illumination at no greater ttian 60 foot candles in die examining room. d) After three minutes take five pupil measurements of 10 seconds duration each wid die PUPILSCREEN assay instrument of each eye for baseline comparison. This will yield 1000 samples of pupil diameter data. e) After completing die baseline readings and saving ti-is data to disk, administer a single drop of die chosen neural transmitter mediator at an appropriate use concentration (e.g. , a .005% tropicamide solution) to one targeted eye chosen arbitrarily. The drop should be administered as described above in step e) of the first preferred protocol. f) After 1 minute have the patient sit up. Wait one minute for die eye to adjust to ambient iUumination and proceed to take five pupil measurements of 6 seconds duration each of die treated eye. Wait approximately 30 seconds between each measurement cycle. g) Repeat me procedure in steps e) and f) widi die other non-treated eye but using a single drop of sterile water for ophthalmic use instead of neural transmitter mediator. h) After administration of the sterile water to the non-treated eye and measurement of me non-treated eye, have the patient wait quietly for a period of five minutes. Repeat the measurements as specified in step f). i) Repeat measurement cycle every 5 minutes up to minute 35 after administration of the tropicamide and sterile water drops. E. Automated Instruments and Systems Suitable for Measuring Pupil Diameter Size A variety of non-invasive automated apparatus is known and commercially available which can be used or modified to meet the minimal operating requirements necessary when practicing the present diagnostic method. Three operational requirements are necessary: (1) continuous monitoring of pupil diameter over time; (2) repetitive measurement of pupil size diameter over a time ranging from less than 1 second to about 5 minutes in duration; and (3) Cumulative recording each measurement of pupil size diameter obtained over time. A number of automated instruments can be used as is or modified to achieve performance of the minimal operating requirements. Examples of such conventional apparatuses are described in U.S. Patent Nos. 4,755,043; 5,187,506; and 4,850,691. In addition, there is a varied class of instruments for measuring pupil diameter which are generally termed "pupillometers." A typical pupillometer measures, displays, and records pupil diameter before and after a light stimulus causes a constriction and redilation of the pupil. These instruments can be modified to eliminate die use of light stimuli to constrict and/or dilate die pupil artificially; and can also be modified to extend the typical manner of usage from making a single measurement to making repetitive measurements over a prechosen time duration in an uninterrupted manner. However, insofar as is known to date, none of these conventional systems has been employed for die diagnosis of Alzheimer's disease; and none of die software and hardware modifications have ever been employed clinically widi such a diagnostic mediod. The primary purpose and essential function of die automated systems is the multiple measurement of pupil diameter size in a serial and repetitive manner to yield a total of at least about sixty and often many thousands individual determinations. The apparatuses described herein-die Series 4000 TV eye movement measurement system and die PUPILSCREEN pupillometers and measurement systems-can determine pupil diameter size every 1/60 or 1/20 of a second; and serially repeat tiiis measurement technique for a short or an extended time period over a desired number of seconds or minutes in duration. Thus, these instruments and systems provide 20-60 individual pupil diameter size determinations per second for as long as deemed necessary or desirable by die user. It is for these reasons that these non-invasive instruments and system are most preferred for use when practicing the present invention. 1. A First Non-Invasive Measuring Apparatus A preferred non-invasive automated apparatus used for performing die diagnostic memodology which is die present invention is die commercially available Series 4000 eye movement measurement system (Applied Science Laboratories, Waltham, Massachusetts). This measurement apparatus and instrumentation was employed widi cognitively intact individuals and widi Alzheimer's disease patients in the experiments described hereinafter; and provides die empirical data presented subsequentiy which illustrate the operability, utility and value of die present diagnostic procedure as a whole. The measurement system employed experimentally is an advanced eye tracker, unobtrusively measuring point of gaze and pupil diameter widi sophisticated data recording and processing capabilities. A TV camera with a telephoto lens (pupil camera) is directed at one of d e subject's eyes. A collimated, near infrared light source tiiat is beamed coaxially with die pupil camera luminates d e eye. The light source is barely visible to the subject as a dim red light. A second TV camera (locating camera) provides a wide angle view of die head to simplify locating me eye. The pupil camera, locating camera, and light source are all enclosed in a single housing called die optical head which can be located up to 230 cm. (90 inches) from die eye. Sixty individual measurements are made each second; and a 30 second viewing duration of die pupil will yield 1800 individual data measurements for die single viewing occasion. The video data is preprocessed, digitized, and sent to an attached computer by me electronics unit as it comes from d e camera 2. A Second Non-invasive Measuring Apparatus Other desirable apparatuses for continuously monitoring pupil diameter size, for repetitively measuring pupil diameter, and cumulatively recording measurement data over time in an uninterrupted manner are die commercially available Pupillometer systems (Applied Science Laboratories, Bedford, Massachusetts). These instruments and systems, in a modified form for clinical use, are well suited for practicing the present memodology to detect Alzheimer's disease in living human subjects. The pupillometer apparatus provides accurate, real-time measurement and display of pupil diameter. The pupil is continuously monitored and pupil diameter is shown directly on a panel meter and also in digital and analog forms. Measurement is also independent of eye movement and other variations over a large field of view. Whereas the TV pupillometer offers accuracy, maximum system flexibility, and high sampling speed, die clinical and field system devices offer simplicity of use, portability, and automatic data recording and display. These devices are ideal for studies primarily concerned widi pupillary reflex function and fast subject output. F. Experiments and Empirical Data To demonstrate die diversity and range of die diagnostic method comprising die present invention, several experimental studies and individual human case history reports are provided below. These experiments and human case history reports are illustrative embodiments of die present invention; and also effectively illustrate die mode and manner in which d e diagnostic metiiod can be practiced to advantage. It will be expressly understood that these experiments and details do not either restrict or limit die present invention in any manner. 1. Experimental Series 1: Hypersensitivity of Alzheimer's Disease Patients a) Subjects A total of 58 individuals were tested for their pupil response to a very dilute solution of tropicamide. These subjects were divided into 5 experimental groups, two patient groups and three groups of elderly controls. The Alzheimer's patient group consisted of 14 subjects who had been previously diagnosed widi probable Alzheimer's disease based on standard clinical criteria. A pilot sample of non-Alzheimer's type dementias consisted of 4 patients widi a diagnosis of Korsakoff s syndrome, multi infarct dementia and dementia widi an extrapyramidal syndrome. Based on neuropsychological screening criteria defined prior to die initiation of die study, 40 elderly subjects were assigned to one of 3 groups. Normal elderly controls consisted of 32 subjects who performed at or above age norms on a battery of neuropsychological tests, tiiat assessed intellectual capacity, attention, memory and language. Five subjects whose performance yielded abnormalities in memory and discrepancies between estimated pre-morbid IQ and current performance in cognitive tests were classified as "suspect" Alzheimer's individuals. Three elderly subjects who exhibited abnormal findings on cognitive tests but had no salient memory deficit were classified as "cognitively abnormal" elderly for this study. The fourteen patients with a diagnosis of probable Alzheimer's disease, males and females, mean age 74 ±7, were drawn from the Massachusetts Alzheimer's Disease Research Center, Boston. Patients widi probable Alzheimer's disease met strict NINCDS- ADRDA diagnostic criteria (see below) for probable Alzheimer's disease, and performed significantly worse than the 32 cognitively normal elderly controls on die Information- Memory-Concentration subtest of the Blessed Dementia Rating Scale, a standard clinical measure of disease severity, (AD = 17 ±7 range = 4 - 27; NC = .7 ± .85 range = 0 - 3; p < 0.01). The scores on die Blessed Dementia Rating Scale range from 0 to 37 with 37 representing die most severe impairment. The NINCDS-ADRDA criteria for a diagnosis of probable Alzheimer's disease require that (i) dementia be established by clinical examination and be documented by a measure such as die Blessed Dementia Rating Scale, (ii) d e patient exhibits deficits in 2 or more areas of cognition, (iii) there is a progressive worsening of memory and other cognitive functions, (iv) die patient has no disturbance of consciousness, (v) there is an onset between die ages of 40 and 90, (vi) there is an absence of systemic or brain diseases that in and of themselves could account for progressive deficits in memory and cognition. Forty-three elderly controls, eitiier spouses of patients or healthy volunteers recruited tiirough advertisement in die metropolitan Boston area were initially enrolled in this study. Three elderly subjects did not meet screening criteria because Of significant ocular pathology (see below) and were not studied. Of die 40 elderly subjects, mean age 72 ±6 (no significant difference from patients), who participated in die study, 32 were considered cognitively normal based on neuropsychological screening. A pilot sample of four patients widi a diagnosis of non-Alzheimer's type dementia were also included for study, mean age 66 ±6. Two of tiiese patients carried a primary diagnosis of Korsakofrs syndrome, one carried a diagnosis of multi-infarct dementia and one a dementia with an extrapyramidal syndrome. All subjects completed an informed consent agreement. Widi die exception of the patient widi die extrapyramidal syndrome (i.e., Pϊirl-i-iisoman-like) all subjects had unremarkable findings on a neuro-ophthalmological examination evaluating saccades, smooth pursuit, visual fields to confrontation and partial field optokinetic nystagmus. No subjects were accepted into the study widi glaucoma, iridectomies or if tiiey were found to have a narrow anterior chamber predisposing diem to closed angle aucoma in response to tropicamide. Three potential normal controls volunteering for this study were not tested due to iridectomies in one or bom eyes. No potential subjects were rejected on die basis of narrow anterior chamber. Normal controls had no diseases of die central nervous system by history. Medication use in patients and all control subjects was comparable. No subject was taking medications widi known interaction effects with tropicamide. Patients were not taking any experimental acetylcholinesterase inhibitors (e.g. Cognex) that might interfere with die assay. b) Preliminary Eye Measurements All subjects were tested at die Laboratory of Eye Movements and Higher Cortical Functions at the Behavioral Neurology Unit at Betii Israel Hospital. Pupil diameter was measured using an Applied Science Laboratories video based, near infrared pupil center to corneal reflection system as described previously herein. This system measures pupil diameter sampled continuously at 60 hertz, is non-invasive, and requires no subject attachments or restraints. For this study, each subject was seated in a comfortable room with dim ambient Ulumination at a distance of 1.5m in front of a TV screen. The subjects' pupils were imaged and measured by die system as they sat viewing the TV screen set at a uniform low Ulumination. Subjects were directed to look at die center of the screen while each of die eyes was individually imaged. Once d e eye was adequately imaged and subjects had ample time to adjust to ambient illumination, a short calibration procedure was performed to insure accurate monitoring of subjects' eye position. Pupil diameter data from die left eye and die right eye of each subject was sampled for 1 minute each to serve as baseline measures of pupil diameter. c) Neural Transmitter Mediator Pupil dilation (mydriasis) of Alzheimer's and control subjects was elicited by die topical application of a cholinergic antagonist, tropicamide, in one targeted eye. Tropicamide, a syntiietic analog of atropine commonly used in ophtiialmology to dilate die pupil and allow examination of die Fundus, takes advantage of die fact diat some of me nerves tiiat control die iris muscle use acetylcholine as a neurotransmitter. Typically, 30 0.5% to 1.0% solutions of tropicamide are used to dilate die pupil maximally in 20 to 40 minutes in normal, healthy subjects. The concentration of tropicamide (0.01 %) used in this study was chosen so as to cause minimal or no dilation of a pupil in a cognitively intact individual, a non- Alzheimer's person. The choice of using a 0.01 % tropicamide concentration for diagnosis determinations was empirically based. Prior to diagnostic testing, experiments with various solution strengths (0.5%-.01 %) were performed to determine die strength concentration that would produce minimal or no dilation (mydriasis) in healthy controls in order to maximize die observed differences in comparison to the AD patient group. Although die entire concentration range experimentally evaluated (0.5%-.01 %) caused some pupil dilation, die higher concentrations caused a maximal dilation rather than a minimal effect. Thus, when tropicamide is employed as die neural transmitter antagomst, die 0.01 % concentration is preferred. d) Protocol and Eye Measurement Procedure After baseline pupil diameter measures were obtained from both eyes, ehher die right or left eye (randomly chosen) was treated widi a single drop of 0.01 % tropicamide solution. Risks to patients and controls were minimized by applying pressure to die inner canthus of the treated eye for 1 min. to reduce systemic absorption of die drug. Pupil diameter data was sampled from die treated eye and de untreated eye individually for 30 sec. durations repeatedly at fixed intervals over die course of an hour as the sampling schedule. Pupil diameter was observed, measured and data obtained at 0, 2, 8, 15, 22, 29, 41 and 53 minutes after administration of tropicamide to one eye; and 30 seconds response was observed and measured at each of these scheduled measurement points-tiaereby yielding 1800 individual pupil diameter measurements per 30 second sampling duration. This yielded a total of seven samples of 30 sec. duration each from the tropicamide treated eye and seven samples of 30 sec. duration each from die untreated eye during die course of die measuring protocol. e) Results The resulting data reveals and unequivocally demonstrates that AD patients have a defect in pupil response compared to normal subjects. This is shown by Figs. 3-6 respectively which provide data from two die experimental groups. Fig. 3 compares the pupil dilation response of patients with clinically determined Alzheimer's disease and experimental controls. As illustrated by Fig. 3, die treated pupils of me normal elderly controls showed a minimal increase in pupil diameter over the course of the hour. In contrast the patients afflicted widi Alzheimer's disease displayed a pronounced response to die pupil dilating effect of tropicamide as shown by the upper curve. Each time point shown in Fig. 3 represents the mean percentage change in pupil diameter over resting pupil diameter (baseline) measurement in the treated eye of Alzheimer's patients and normal elderly controls. A Kurskal Wallis pairwise multisample test was used to determine die significance of d e differential tropicamide sensitivity of die Alzheimer's and control groups. Overall die results indicated tiiat at minute 29 mere is a 23.4% (SEM 3.8%) change in d e pupil diameter of patients widi probable Alzheimer's disease compared to a 5% (SEM 1.7%) change for normal elderly subjects (p= .009). Fig. 4 compares die response of patients widi clinically determined Alzheimer's disease, the suspect Alzheimer's individuals, d e cognitively abnormal elderly subjects, die patients with non-Alzheimer's type dementia, and die normal controls. As shown by Fig. 4, d e percentage change in pupil diameter of die treated eye over baseline of die suspect Alzheimer's subjects and d e cognitively abnormal elderly closely parallels that of the patients widi probable Alzheimer's disease, while die patients widi non- Alzheimer's type dementia exhibit a pattern like that of normal elderly controls. Both die suspect Alzheimer's disease individuals and die cognitively abnormal elderly subjects show an almost identical pattern of pupillary response to that of patients widi clinically determined Alzheimer's disease. In contrast, die response of die group of patients diagnosed widi non- Alzheimer's type dementia is similar to that of normal elderly controls. The complete set of data for die 29 minute sampling point (die point of maximal separation of clinically determined Alzheimer's patients and normal elderly control subjects) is presented by Fig. 5. Each symbol represents die percent change in pupil size over baseline of a single individual. Fig. 6 plots de mean scores and die ±95% confidence intervals of die means for patients with probable Alzheimer's disease and die normal elderly controls. There is a clear separation between tiwse groups beginning at minute 15. This distinct separation between die groups is maintained at minute 29 after instillation for the ±99% confidence intervals (not shown). Note that a minimum overlap in the pupil dilation scores between groups and between individual subjects in different groups is obtained by designating 13% change in pupil diameter at minute 29 of die assay as a cutoff point. Of die 40 elderly subjects from the community tiiat were tested, (normal elderly controls (NC), suspect Alzheimer's dementia individuals (SAD), and cognitively abnormal elderly subjects (CAE)), 9 showed a positive response to die assay that was greater than or equal to 13% at minute 29, of which 7 were either in the suspect Alzheimer's disease group or die cognitively abnormal elderly group. Thus, only 2 of 32 normal elderly controls exhibited an exaggerated positive pupil response to the assay, but had no other clinically notable cognitive or neurological defects. This number of positive pupil responses in our "normal" elderly sample is within die order of magnitude one should expect from previous studies of die prevalence of this disease in the community. It is therefore believed that tiiese 2 individuals may have sufficient Alzheimer's pathology to register a positive pupil finding, but do not yet exhibit clinically discernible symptoms of cognitive decline. Of d e 4 patients widi non-Alzheimer's type dementia (NAD), included as a pilot sample, 3 showed a minimal response to the pupil assay and reacted as d e normal elderly sample. One subject (diagnosed widi Korsokoff s syndrome) exhibited a pupil response similar to that of patients widi probable Alzheimer's disease. These data indicate tiiat, with few exceptions, botii patients with a diagnosis of probable Alzheimer's disease and die subjects we have classified as "suspect" Alzheimer's individuals can be distinguished from d e normal elderly controls on die basis of their hypersensitivity to tropicamide. Furthermore, die fact tiiat die response of patients in the pilot sample with non-Alzheimer's type dementias is similar to that of normal elderly controls, shows that the pupil dilation assay is specific for Alzheimer's padiology. When die data from the 14 patients is combined wid probable Alzheimer's disease and die 5 subjects termed suspect Alzheimer's individuals, 18 of 19 exhibited a positive response to the pupil dilation assay. This 95% concordance between die clinical or suspected diagnosis and die results of die pupil assay is consistent widi die finding in our dementia clinic tiiat 95% of patients who are clinically diagnosed widi probable Alzheimer's disease and who are subsequently brought to autopsy have pathologically confirmed Alzheimer's disease. f) Conclusions Several findings from this study demonstrate that the pupil dilation test is able to identify Alzheimer's patients prior to the onset of clinical symptoms of dementia. First, patients with a clinical diagnosis of Alzheimer's disease who exhibited an exaggerated mydriatic response included die most mildly demented individuals as measured by die Information-Concentration-Memory subtest of the Blessed Dementia Rating Scale and no correlation was found between patients' dementia scores and a positive pupil result. The lack of such a correlation suggests that the pupil assay may be sensitive to the earliest stages of the disease. Secondly, almost all elderly subjects living in the community who were tested and who showed a positive pupil response also exhibited neuropsychological deficits and most were found to have a salient memory impairment consistent with Alzheimer's disease. Of particular interest is the case of patient SG (described in detail subsequently by Case Study 3). This elderly subject living in die community initially exhibited a positive pupil response to the test methodology but showed no obvious cognitive deficits and only a self report of mild difficulty wid some daily living activities. He was re-tested nine months later and continued to show a positive pupil response. During this interval, patient SG exhibited a substantial decline (from 0 to 6) on die ormation-Concentration-Memory subtest the Blessed Dementia Rating Scale and developed clear memory deficits. These results indicate that die pupil response assay was sensitive enough to detect an abnormal response in an elderly community-dwelling individual who subsequently developed symptoms consistent with a diagnosis of probable Alzheimer's disease. In sum, patients with clinically determined Alzheimer's disease can be distinguished from normal elderly subjects and patients widi non-Alzheimer's type dementia on die basis of pupil diameter changes induced by a dilute solution of a cholinergic antagonist applied to die eye. All but one of die patients widi probable Alzheimer's disease showed a pronounced hypersensitivity to a neural transmitter mediator, tropicamide; and tiiere were only 2 of 32 subjects in the normal elderly sample who exhibited a response similar to tiiat of the Alzheimer's patients. Overall die findings from this experiment series show tiiat with respect to normal elderly controls and patients with probable or clinically suspect Alzheimer's disease the pupil dilation assay has a sensitivity of 95% and a specificity of 94%. Unlike other biochemical and physiological tests now being developed, the pupil 34 response test is safe, relatively non-invasive, sensitive, and easy to quantitate with already- available automated instrumentation. 2. Experimental Series 2: Human Case Studies a) Case 1 : Patient Widi Probable Alzheimer's Disease AA is a 77 year-old woman, retired educator widi a 5 year history of an insidiously progressive decline in mental state functioning which has left her increasingly dependent on her daughter to supervise her daily living activities. Her initial symptoms involved forgetfulness and this has subsequently gotten increasingly worse. Currently, she cannot recall events that have occurred 5 minutes before. Neuropsychological testing revealed a prominent amnesic syndrome. She had preserved attention, but had marked difficulties storing or retrieving new information and exhibited a rapid rate of forgetting. She also had a mild anomic aphasia. She scored a 14/37 on d e -hformation-Memory-Concentration (IMC) Subtest of the Blessed Dementia Scale (BDS), which is in the mild-moderate range of severity. (A score of 37 is die worst a patient can do.) Her score on die left side of die BDS tiiat evaluates daily living activities, habits, personality, and behavior was 8/28, consistent with mildly compromised functioning. (A score of 28 would represent die most impaired status.) Elementary neurological examination was within normal limits. Cranial magnetic resonance imaging (MRI) was unremarkable for her age. EEG showed non¬ specific slowing. Metabolic studies were within normal limits. Her history, neurological examination, and pattern of neuropsychological deficits as well as her unremarkable work-up yielded die diagnosis of probable Alzheimer's disease and was consistent with NINCDS-ADRDA criteria. She exhibited a hypersensitive mydriatic response to testing via the preferred protocol with tropicamide. b) Case 2: Patient With Probable Alzheimer's Disease BB is a 66 year-old retired housekeeper widi an eighth grade education who exhibited 2 χ years of progressive cognitive decline. Initially tiiere were occasional episodes of forgetting events or activities as well as noticeable word finding pauses. Currently, she has major difficulties remembering daily events, conversations and dungs that she may have read or seen on television. BB is requiring increasing supervision to manage her daily living activities. Neuropsychological testing revealed mild attentional difficulties widi substantial problems storing and retrieving infoπnation after brief delay and a marked anomia. BB scored a 11/37 on die IMC Subtest of the BDS. Elementary neurological examination was unremarkable. MRI was within normal limits for age. BB's clinical history, pattern of neuropsychological impairments, and negative work-up led to the diagnosis of probable Alzheimer's disease. BB exhibited a hypersensitive mydriatic response to testing via the preferred protocol. c) Case 3: Elderly Subject Widi "Suspect" Alzheimer's Disease SG is a 79 year-old retired male executive, with a BA from Harvard College, who volunteered to participate in our study. During his initial test session he shared concerns about possible changes in his cognitive abilities. Clinically, it was suspected that he may be exhibiting very subtle problems with his cognitive and functional status. He scored a 6/28 on die left side of the BDS, suggestive of mild changes in his daily living activities. However, his score on the IMC Subtest of the BDS was 0/37, which clearly was within normal limits. On the preferred protocol tropicamide test, his pupil exhibited a hypersensitive response. He agreed to a follow-up evaluation. Nine months later the subject was retested. He continued to show mild changes in his activities and interests (scoring an 8/28 on die left side of die BDS). His performance on the IMC Subtest of die BDS was now 6/37, consistent widi mild cognitive impairment. Further neuropsychological testing revealed an estimated premorbid 10 in die 93 percentile (FSIQ of approximately 123). However, his performance on die Boston Naming Test was only low average and he exhibited clear memory problems, performing much below his estimated premorbid capacities. There was a marked loss of newly learned information after a delay period. Elementary neurological examination was unremarkable. These results suggested an early dementing illness, with a salient amnestic component. This neuropsychological pattern was consistent with the diagnosis of probable Alzheimer's disease. d) Case 4: Elderly Subject widi Suspect Alzheimer's Disease HH is a right handed 78 year-old retired engineering executive with 17 years of education living with his wife in die community, who volunteered as a subject in a study of aging and Alzheimer's disease. The subject had an unremarkable medical history with no report of diabetes, hypertension, seizure or stroke. His background is remarkable for a father who had clinically diagnosed Alzheimer's disease in his early eighties. HH had an unremarkable neurological examination. His estimated pre-morbid IQ was 125. His performance on the Information-Memory-Concentration subtest of the Blessed Dementia Rating Scale was 0/37 with 0-3/37 being considered normal. Naming was unremarkable for age. However testing of attention revealed some mild impersistence. Memory performance appeared to be mildly impaired as tested on immediate and delayed recall on die verbal and visual portions of d e Wechsler Memory Scale. HH encoded information adequately but lost information on delayed recall. On delayed visual recall he scored only at die 29th percentile for age which is inconsistent with his estimated premorbid IQ. HH was sent for a SPECT scan which revealed perfusions defects in inferior temporal and superior parietal corτicies consistent with the SPECT pattern seen in Alzheimer's patients. The pattern of findings widi this subject is consistent with mild changes in memory and attention. On the preferred pupil protocol, HH exhibited a positive response. He was classified as a suspect Alzheimer's patient. e) Case 5: Patient with Dementia of die Non- Alzheimer's Type BB is a 79 year-old priest with a Ph.D. in religious studies, who presented widi an atypical Parkinsonian syndrome not responsive to Sinemet. His motor disabilities were characterized by bradykinesia, a "masked" face, hypophonia, micrographia, and slowed gait. These symptoms have been slowly progressive and are consistent with a degenerative disease affecting die basal ganglia and its connections. Over the past 4 years, he has exhibited progressive and marked decline in mental abilities. Initially, he scored a 2 on die IMC Subtest of the BDS. Four years later, he scored a 7. He has exhibited increasingly slowed cognitive processing speed, impaired complex attention, and memory difficulties due to limited attention span. Primary memory abilities (i.e. retention) and basic reasoning skills have been relatively well preserved (e.g. performing at the 98th percentile on die Ravens Progressive Matrices Test). His cranial MRI was unremarkable. The patient's history and pattern of cognitive decline is noj consistent wid probable Alzheimer's disease. Rather, it suggests a dementing illness primarily affecting the frontal networks and their subcortical connections, sometimes referred to in die literature as a "subcortical dementia" . BB's pupil dilation response to testing using the preferred protocol was within die normal range. f) Case 6: Normal Elderly Control Subject CC is an 87 year-old women who is living independently in die community and managing all of her daily activities and financial matters. She retired 20 years ago at age 67 and has remained socially active in her community. Past medical history was very benign, widi no report of hypertension, diabetes, or strokes. She only scored 0.5/28 on the left-side of die BDS illustrating no significant alterations in activities, behavior, or personality. On the IMC Subtest of the BDS, she scored a 2/37 which is witiiin normal limits. Her estimated IQ was 121. Concentration and naming were unremarkable for her age. Immediate and delayed recall on the verbal and visual portions of die Wechsler Memory Scale were performed at die 80-86tiι percentile for elderly individuals. Her elementary neurological examination was benign. In summary, based on her current neurological, neuropsychological, and functional status, there is no evidence tiiat she is suffering from a dementing illness and she fits all criteria for being a "normal" elderly control subject. Upon testing via the preferred protocol wid d e pupil response assay, she did not exhibit a hypersensitive response. Section II: Diagnosis of Alzheimer's Disease Based Upon Light-Stimulated Pupil Constriction Velocity A. The Hypersensitive Pupillary Dynamic Responses If and when a person suspected of having Alzheimer's disease is clinically examined, die pupils of die subject typically appear to be similar to those who are cognitively normal. The pupils of the Alzheimer's patient may be examined for size, shape, near response, and consensual light reaction without demonstrating any major defect. Light directed into one pupil will typically result in normal constriction of the pupils in both eyes. As widi any population of individuals, some person's pupils may be markedly constricted; others may have unequal pupils: and die visual acuity of the person may be normal, show distant vision, or be near sighted. No casually observed feature of die pupil alone therefore can provide a basis for making a differential diagnosis. In contrast, die hypersensitive dynamic responses of die pupil in the Alzheimer's disease patient present an observable, reproducible and reliable basis for clinical diagnosis. The pupils of Alzheimer's afflicted subjects respond abnormally to unusually small or dilute concentrations of exogenous neural transmitter mediators (cholinergic antagonists and agonists) intentionally introduced to die eye. Equally important, the pupil of die Alzheimer's patient responds to concentrations of neural transmitter modulators which cause little or no response in cognitively normal persons. A range and variety of hypersensitive pupillary responses are individually identifiable and measurable in d e Alzheimer's disease patient. For example, as described above in section I, the pupillary hypersensitive reaction can manifest itself as an abnormal mydriatic response to an unusually small concentration of an anticholinergic agent (such as 0.01 % tropicamide); or as a miotic response to an unusually dilute concentration of a cholinergic agonist (such as 0.01 % pilocarpine). In these examples, the hypersensitive pupillary response can be repeatedly observed and quantitatively measured (without any photostimulation) by determining pupil dilation or pupil contraction. The method described in die present section employs an entirely different manifestation of the hypersensitive pupillary response. The hypersensitive manifestation observed, quantitatively measured, and utilized as die test parameter is the change in constriction velocity for the pupil in response to stimulation by visible light as a consequence of introducing a dilute cholinergic antagonist to die eye. The concentration of exogenous neural transmitter mediator (die cholinergic antagomst) administered to the eye of die subject is chosen so as to be insufficient to cause any marked pharmacological or physiological change in a cognitively intact normal person but is adequate to induce a substantive change in the pupil constriction velocity of die hypersensitive Alzheimer's disease patient. The manifestation thus identifying and distinguishing die Alzheimer's subject is die significant change in the constriction velocity of pupil response to photostimulation in comparison to cognitively normal persons receiving the same concentration of neural transmitter mediator. B. The Essential Parts Of The Methodology There are four essential requirements for practicing the present diagnostic method, each of which may be satisfied widi a variety of articles or procedures: (1) The method requires the use of non-invasive means for introducing photostimulating visible light of predetermined wavelength and intensity to die eye sufficient to cause a constriction of the pupil; and for determining die velocity of die pupil constriction initiated by die photostimulation. A variety of preferred automated apparatuses and systems are described herein but die memodology as a whole is not dependent upon any specific apparams, instrumentation, electronics, circuitry, optics, or system. Accordingly, any means which individually or integrally provides die requisite eye photostimulation and constriction velocity determination will suffice for purposes of practicing the present invention. (2) The method requires the administration of at least one neural transmitter mediator to an eye of the person undergoing diagnostic testing. This mediator is a compound selected from the group consisting of cholinergic antagonists. Moreover, the quantity and concentration of this neural transmitter mediator must be sufficiently dilute such that a cogmtively normal person receiving this mediator will not show or reveal a marked or substantive change in his constriction velocity after photostimulation. (3) The method requires that at least one eye of die person undergoing diagnostic testing be subjected to stimulating visible light energy after administration of die neural transmitter mediator to that eye. The range of visible light wavelengths, visible light intensities, time duration of visible light stimulation, and frequency of repeated visible light stimulation may be varied. Accordingly, d e substantive requirement is only that the eye of die test subject be stimulated by visible light after die introduction of a neural transmitter mediator. (4) The metiiod requires that die constriction velocity after photostimulation be determined after die neural transmitter mediator has been administered to die eye. This constriction velocity deteπnination can be made using any article, machine, measurement system, method of calculation, and display mode conventionally known or commercially available. C. The Diagnostic Method 1. The Neural Transmitter Mediator: The present diagnostic methodology utilizes die constriction velocity of die pupil in response to stimulation by light as the essential diagnostic feature. In order to utilize pupil constriction velocity as a parameter (rather than any odier type of pupillary dynamic change) one must administer a dilute concentration of a cholinergic antagonist. A representative, but non-exhaustive listing is provided by Table 4 below. Table 4: Exogenous Neural Transmitter Mediators Anticholinergic Agents (cholinergic antagonists) Name Brand Conventionally Used Present Use/ Agent-Generic Example Doses Comments Tropicamide Mydriacyl 0.5-1.0% Usually 1.0% Atropine Atropisol 1 % Not routinely used for eye examinations in adults Homotropine I-Homatrine 2% q 10-15 min Used for Hydrobromide refraction not dilation Cyclopentolate Cyclogyl 0.5-2% 0.5% for Hydrochloride fundoscopic examination Scopolamine Isopto Hyoscine 0.2-0.25% Used for post-op mydriasis not eye examinations It will be recognized and generally understood by a person ordinarily skilled in the anatomy of the eye that die pupil is formed by die muscles and pigmented stroma of the anterior uveal tract (the iris). There are two types of muscles: a circumferential sphincter found in die margin of the iris, innervated by d e parasympathetic nervous system and radial dilator muscles which run from d e iris margin to e root of die iris, innervated by die sympathetic nervous system. Pupil size represents a balance between stimulation from the parasympathetic and sympathetic nervous systems. Constriction of the pupil (miosis) is caused by the stimulation of the parasympathetic fibers, whereas dilation (mydriasis) is caused by sympatiietic activation. These systems generally contain neurons tiiat are driven by cholinergic or adrenergic neurotransmitters respectively. The neuro-physiology of me pupil and iris make it an ideal physiological marker for measuring the integrity of cranial nerve, midbrain and central nervous system functions. 2. Measurement Parameters And Procedural General Guidelines A range of general procedural guidelines and measurement parameters are provided herein for the optimization and convenience of both die user and d e individual being tested. These general procedural guidelines are provided for the benefit and advantage of die intended user; and die measurement parameters are merely illustrative possibilities, examples and suggestions to consider and use when preparing detailed protocols intended for use on a clinical basis. 3. The Repetitive Cycle and die Sampling Occasion An essential part of die present methodology is die use of a non-invasive automated apparams to monitor pupillary dynamic changes and to determine constriction velocity (the rate of pupil size changes) after stimulation by a known quantity of visible light energy. Each observation and individual determination of constriction velocity for the pupil constitutes one measurement "episode" or "epoch"; and a measurement episode is normally performed in less than seconds, e.g., die normal period of pupil constriction in response to photostimulation, and, further, is performed repetitively and cyclically. The preferred automated apparams is able to monitor and measure pupil diameter size change repeatedly- both before and after photostimulation continuously at a rate of about 60 pupil measurements per second. 4. Frequency of Sampling Occasions It is desirable that at least two sampling occasions separated by a prechosen length of time be made when practicing die present diagnostic method. The first sampling occasion constitutes die "zero" time and provides die initial baseline characteristics of untreated pupil constriction velocity for tiiat individual patient. It is expected and intended that tins initial baseline sampling occasion be made on botii die left eye and die right eye of die patient. One eye, randomly chosen, will be d e eye treated with the neurotransmitter mediator; and die otiier eye will be treated widi a non-drug control solution. The present diagnostic method and protocol demands that at least a second sampling occasion be performed after administration of the neural transmitter mediator to die targeted eye, preferably when die maximum change and difference in pupillary dynamic response occurs. The methodology preferably employs minimally two different sampling occasions during which the constriction velocity of the treated targeted eye and also of die non-treated control eye are measured. In better protocols, from 3 to about 6 different sampling occasions are performed after introduction of die neural transmitter mediator over a period of about one hour. This greater sampling will lead to results which more accurately identify die hypersensitivity of die pupillary response and die greatest change and maximal effects of treating the targeted eye widi die chosen neural transmitter mediator. Thus, the preferred protocol will have 6 different sampling occasions of at least 5 measurement episodes each. 5. Concentration of the Chosen Neural Transmitter Mediator Preferred embodiments will employ concentrations of neural transmitter mediator which does not cause significant changes in pupil constriction velocity vs. baseline prior to pharmacological treatment (meaningful changes in the rate of change for pupil diameter size) after photostimulation in individuals widiout Alzheimer's disease. In addition, die concentration should be high enough so that Alzheimer's disease patients show a marked hypersensitivity in pupillary response to photostimulation. Accordingly, neural transmitters and concentrations are, in general, determined and employed in die same manner as set forth in Section I. 6. Constriction Velocity Determination Cycles The calculation of pupil constriction velocity is made using pupil diameter size data obtained both before and after photostimulation. Each cycle of repetitious measurement includes a first pupil diameter size determination, followed by photostimulation, and then a measurement of die stimulated (constricting) pupil. The pupil is then allowed sufficient time to re-dilate to its original diameter size state; and another measurement cycle to determine constriction velocity is then initiated. The preferred automated instruments described hereinafter can perform 5-60 pupil size measurements per second. The initial and follow-up measurements of pupil size in each measurement episode are preferably made using near infrared light; and employ botii die apparatuses and procedures described witiiin U.S. Patent Nos. 4,755,043 and 5,187,506, die texts of which are expressed incorporated by reference herein. Sources providing wavelengdis of about 800 nm-2000 nm are used; and wavelengths from about 850 nm to 900 nm are deemed best. The light intensity is adjustable and preferably lies in die range of 1.5-6.5 mw/cm 2 . 7. Basis For Comparing the Empirically Obtained Data in Order to Diagnose Alzheimer's Disease A diagnosis is preferably made when die pupil constriction velocity of the eye treated widi either a cholinergic antagonist or agonist changes substantially and is significantly different than a predetermined range of numerical values representative of the cognitively intact population as a whole. The difference from d e normal standard range of numerical values is considered die diagnostic criterion for determining the presence or absence of Alzheimer's disease in a living human individual. This diagnostic evaluation is empirically determined by examining the percentage change in pupil constriction velocity of known Alzheimer's patients and of lmg jl cogmtively intact individuals to a particular neural transmitter substance at a particular concentration; and determining die point at which known Alzheimer's patients compared to known cognitively intact individuals are separable and distinguishable in the magnitude of tiieir response. D. Preferred Protocol The present diagnostic method employs non-invasive automated apparatuses and systems which can observe and repetitively determine pupil constriction velocity over short time intervals in an uninterrupted manner. The manner of observation and repetitious measurement yields constriction velocity determinations of from about 5-60 measurements per sampling occasion. Different automated systems may vary in their speed of measuring pupil size changes, and die time duration of each sampling occasion may be extended or shortened accordingly. The preferred protocol presented below is illustrative of the diagnostic methodology as a whole. The preferred protocol is intended to accommodate die different automated equipment and clinical circumstances in which persons suspected of being afflicted widi Alzheimer's disease are to be encountered. The preferred protocol is as follows: a) Prior to administering the pupil assay, the following patient screening tests must be done: 1. Evaluate d e patient for any ocular abnormalities: a. cataracts. b. history of glaucoma. c. a narrow anterior chamber. d. local corneal pathology that might affect corneal permeability (e.g. , dry eye or poor tear lakes). 2. If patient exhibits condition "b, c, or d" do not proceed widi die test. 3. If patient has cataracts that distort die shape of die pupil excessively do not administer die neural transmitter mediator to the affected eye. 4. Screen patients for any current use of drugs widi central or peripheral cholinergic effects. If patient is currently using medications with known cholinergic effects, note on die patient record for future reference in interpreting pupil assay data. b) Once screening has been done, insure that die patient is alert and not agitated or drowsy, If patient is excessively drowsy do not proceed widi test, but schedule die patient for future testing, c) Allow five minutes for patient to sit quietiy while pupils adjust to ambient photopic iUumination at no greater than 5 foot candles in the examining room. d) After five minutes, image die patient's eye with a 1050 Pupillometer eye measurement system. Set die pupil discriminator such that the eye is completely encircled widi die white discriminator and forms a clean elliptical image in the center of the pupil monitor. Open a data file and begin recording pupil diameter data. Stimulate die eye by means of die photostimulator and continue to record data. Repeat this process after five seconds rest each time for an additional five measurement episodes. Repeat die entire procedure for d e untreated eye. e) After completing de baseline readings and saving this data to a file, -idminister a single drop of die chosen neural transmitter mediator in die appropriate concentration (e.g. , a 0.01 % tropicamide solution) to one targeted eye chosen arbitrarily. The drop should be administered in me following manner: 1. Have the patient in a position on a chair or an examining table so that they can tilt their head well back. 2. Hold open die lower and upper eyelid wid die thumb and first finger. 3. Squeeze the bottle of treatment solution gently so as to allow a single drop to fall on the center of die lens of die eye. 4. Have die patient close his/her eye after administration of die drop. 5. Administer gentle pressure on the inner canthus of the eye for 1 minute to prevent excessive entry into die systemic circulation. 0 After 1 minute have the patient sit up. Wait one minute for the eye to adjust to ambient illumination and proceed to image die pupil as described in (d) above. Record 5 seconds of pupil constriction velocity deterrninations separated by 30 second intervals to a data file, g) Repeat the procedure in steps (e) and (f) with the otiier non-treated eye but using a single drop of sterile water for ophthalmic use. h) After administration of the sterile water drop to die non-treated eye and measurement of die non-treated pupil have d e patient wait quietiy for a period of 5 minutes, i) After 5 minutes have elapsed, wait for 1 minute while die patient's eye accommodates to die low Ulumination. Proceed to image the eye again as described in step (f). Record five constriction velocity measures from the treated eye. Repeat this procedure with die untreated eye. j) Repeat the pupil constriction velocity determination procedure every 5 minutes until about minute 30 of the test, k) After the last reading at about test minute 30, have die patient wait for 10 minutes more. After 10 minutes, again record pupil constriction velocity determinations from die treated and untreated eyes as described above. 1) Have die patient wait a final 10 minute segment and then record pupil constriction velocity determinations from the treated and untreated eyes as described above. The final reading should be taken approximately 55 minutes after administration of the eye drops. E. Automated Instruments and Systems Suitable For Measuring Pupil Constriction Velocity. A variety of non-invasive automated apparatuses are known and commercially available which can be used as is or modified quickly to meet die minimal operating requirements necessary for practicing the present diagnostic metiiod. Examples of such conventional apparatuses are described in U.S. Patent Nos. 4,755,043, 5,187,506; and 4,850,691. In addition, the "pupillometers" described above in section I may be used. The apparatuses described below can determine pupil constriction velocity every 1-3 seconds; and repeat this cyclically for a short or an extended time period. 1. First Non-invasive Apparams: A Photostimulator/Controller In Combination Witii A TV Pupillometer The Series 1000 Photostimulator and Controller is a powerful device for tests involving the introduction light pulses to one or both eyes of a subject. The beams of light are controllable in exposure frequency, pulse width, focus, beam diameter and intensity. For pupillometry, the Photostimulator/Controller may be used widi die companion Applied Science Laboratories Series 1050 TV Pupillometer. One or both eyes may be light stimulated; and die controls for die two eye channels may be synchronized in any desired phase and temporal relationship in order to test binocular responses. The controller provides a very convenient way of programming the shutter exposure times for one or two channels. The pulse width and de period may be controlled for each channel, and die phase relationship between the two systems can also be determined. This provides virtually any pulse profile that may be desired. Continuous cyclic operation or single pulse actuation is possible. The two channels may be locked phase or randomly related. The Model 1050 TV pupillometer provides accurate, real-time measurement and display of pupil diameter. The pupil is continuously monitored; pupil diameter is shown directly on a panel meter; and pupil diameter is shown in digital and analog forms. Pupil diameter measurement is independent of eye movement and other variations over a large field of view. The TV pupillometer uses a near infrared illuminator and a low light level, solid state CCD television camera to observe the eye. A pupil recognition circuit automatically distinguishes the pupil from the iris, the eyelids, and otiier noise wid minimum operator adjustment. A television monitor displays die image of the eye widi superimposed pupil delimiters to clearly indicate die accuracy of die measurement. The automatic circuitry will maintain proper measurement for a large range of settings and conditions. In operation, die subject's head is usually stabilized by a chin rest or a chair with a headrest. There must be an unobstructed visual path to die eye. The operator then adjusts die optics and monitor to obtain a clear image of die subject's eye. Afterwards, die operator adjusts die discriminator control until a crescent appears at the left edge of die pupil and delimiters appear in the monitor above and below die pupil. As long as the delimiters are properly positioned, the measurement of pupil diameter is correct, in spite of any other noise or artifacts. Pupil diameter in millimeters is displayed on a panel meter and provided as analog and digital signals. Pupil diameter size is usually measured vertically; however, horizontal diameter and pupil area measurement are also possible. 2. Second Non-Invasive Apparams: the PUPILSCAN System Whereas Model 1050 offers accuracy, maximum system flexibility, and high sampling speed, die S-6 and S-7 devices offer simplicity of use, portability, and automatic data recording and display. The S-6 and S-7 devices are ideal for clinical or field studies primarily concerned with pupillary reflex function and fast subject throughput. The S-6 device, also known as the PUPILSCAN™ apparatus, is a binocular tabletop device. Both devices are interfaced to any IBM compatible PC via a conventionally supplied interface board and cable.. When die trigger position operating switch an die S-6 optical unit is depressed, infra-red illumination is turned on and a reflected image of die pupil is focused on an electronic image sensor. The Ulumination is adjusted automatically by the program to d e optimum level simplifying use under widely variable ambient light conditions. To aid in centering the instrument on the pupU, a pair of red diodes on d e cross hair ring are illuminated or extinguished when satisfactory image position has been achieved as a signal to the operator to release the operating switch to make the measurement. When the switch is released, die program fine tunes infrared illumination and automatically fires selectable intensity green diodes for a programmed duration stimulus pulse. Maximum velocity of constriction is displayed in millimeter per second and die amplitude of pupil constriction is calculated and displayed. In addition, a time plot or pupU response curve appears automatically at the end of each measurement cycle. The plot for subsequent determinations replaces the previous curve and digital data are added beneath those of earlier cycles for easy comparison of successive data measurements. PupU constriction velocity vs time plots may be printed directiy without leaving die measurement mode or at the conclusion of a set of measurements. Saved measurement data may be recalled and additional measurements may be added to the file allowing comparison of current data widi measurements from an earlier session, creation of a cumulative patient history, etc. 3. A Third Non-Invasive Apparatus: The PUPILSCREEN System The S-7 device or PUPILSCREEN™ instrument is designed for convenient binocular measurements. A knob on the device quickly alternates the measurement from left to right eye. The PUPILSCREEN™ devices are designed to automatically display pupil images and pupillary reflex graphs on a computer screen. Computer files are created which can be further manipulated by user programs or the optional spreadsheet analysis templates which we offer. The S-7 instrument assembly functions on a plug-in accessory to an IBM PC or compatible personal computer; is operated by an easy-to-use, menu-driven, program, offering a range of programmable measurement variables; and provides for automatic storage of pupil measurement data as well as retrieval and analysis of subject data from a database for rapid comparisons widi previous or baseline measurements. The S-7 device is a table-top instrument ideal for large volume screening tests in which die subject aligns his eyes with aid of a video image of die pupU. Once aligned, d e subject himself presses a switch to initiate an automatic single or multiple cycle measurement sequence. Once the eye to be measured is set by rotating the selector knob on die top of de unit, subject identification is entered from die computer keyboard in response to a screen prompt. The subject positions his head against the foam rubber face pad and fixes his gaze straight ahead where die pupU image will be displayed in die optical unit. The subject then initiates the measure cycle by pressing a switch. After each measurement cycle die computer monitor will display die pupU response curve and die key parameters of pupU size and response characteristics wUl appear on the monitor display. Each additional measurement cycle wUl be recorded and displayed independently on die monitor.

Claims: Claims 1. A non-invasive method for diagnosing the presence or absence of Alzheimer's disease in a living subject which comprises: adiTiinistering to one of said subject's eyes at least one neural transmitter mediator in an amount insufficient to cause a significant pupil constriction or dilation if said subject is not afflicted with Alzheimer's disease, repetitively and episodically measuring pupil diameter in said treated eye during at least a part of the time said neural transmitter mediator or mediators would have an observable effect on pupil diameter in a subject afflicted with Alzheimer's disease, and diagnosing the presence or absence of Alzheimer's disease in said subject based on the presence or absence of Alzheimer-characteristic pupil diameter changes from a baseline pupil diameter established for said treated eye or Alzheimer's-characteristic pupil diameter rates of change calculated from said pupil diameter change measurements. 2. A method according to claim 1, which further comprises: photostimulating said subject's treated eye with one or more episodes of visible light to induce pupillary constriction, such that said episodic measurements of pupil diameter occur during the pupil contriction in response to said photostimulation, said pupil diameter measurements being thereafter used to determine the rate of change of pupil constriction. 3. A method according to claim 1 wherein said repetitive and episodic measurements comprise at least 1000 measurements per episode of measurement. 4. A method according to claim 2 wherein said repetitive and episodic measurements comprise measurement of at least 50 Hertz during said constriction. 5. A method according to claim 3 or 4 wherein at least three episodes of measurement are taken. 6. A method according to claim 3 or 4 wherein said eposides of measurements extend timewise to at least a period of expected maximal pupillary response. 7. A method according to claim 1 wherein said neural transmitter mediator is a cholinergic antagonist. 8. A method according to claim 7 wherein said cholinergic antagonist is about 0.01 % tropicamide. 9. A method accorrding to claim 8 wherein said repetitive and episodic measurements comprise measurements at 60 Hertz over 30 seconds for each such episode and wherein at least five such episodes extend over at least 30 minutes. 10. A method according to claim 10 wherein the Alzheimer' s-charactistic change in pupil diameter is at least a 13% change from the normal control group. 11. A non-invasive method for diagnosing Alzheimer's disease in a living human subject, said diagnostic method comprising the steps of: providing a non-invasive automated apparatus which can continuously monitor pupU diameter size over time, repetitively measure pupU diameter size over time for a prechosen duration ranging from about less than 1 second to about 5 minutes, and cumulatively record such monitored and measured pupU diameter size information as is obtained over time; identifying one eye in die living human subject as a targeted eye; using said non-invasive automated apparatus on a first measurement occasion to continuously monitor, repetitively measure, and cumulatively record die pupU diameter size of said targeted eye in the living human subject over the prechosen duration to provide primary information of pupU diameter size for said targeted eye; administering at least one neural transmitter mediator to said targeted eye of die living human subject in an amount insufficient to cause marked dUation of pupU diameter over time in a person not afflicted with Alzheimer's disease, said neural transmitter mediator being selected from die group consisting of cholinergic antagonists and adrenergic agonists; waiting a predeter-i-rined interval of time for said administered neural transmitter mediator to act upon said targeted eye; then using said non-invasive automated apparams on at least a second measurement occasion to continuously monitor, repetitively measure, and cumulatively record die pupU diameter size of said targeted eye in the living human subject over the prechosen duration to provide secondary information of pupU diameter size for said targeted eye after being acted upon by said administered neural transmitter mediator; and determining such change in pupU diameter size for said targeted eye as may have occurred over said time interval by comparing said primary information widi at least said secondary information, a marked increase in pupil diameter size for said targeted eye diagnostically establishing that living human subject as being afflicted with Alzheimer's disease. 12. The diagnostic method as recited in claim 11 wherein said neural transmitter mediator is an agent selected from the group consisting of tropicamide, atropine, homotropine hydrobromide, cyclopentolate hydrochloride, and scopolamine. 13. The diagnostic method as recited in claim 11 wherein said neural transmitter mediator is an agent selected from die group consisting of phenylephrine hydrochloride, hydroxyamphetamine hydrobromide, dipivefrin, and epinephrine. 14. The diagnostic metiiod as recited in claim 11 wherein said neural transmitter mediator is a combination of cyclopentolate hydrochloride and phenylephrine. 15. A non-invasive method for diagnosing Alzheimer's disease in a living human subject, said diagnostic method comprising die steps of: providing a non-invasive automated apparatus which can continuously monitor pupU diameter size over time, repetitively measure pupU diameter size over time for a prechosen duration ranging from about less than 1 second to about 5 minutes, and cumulatively record such monitored and measured pupU diameter size information as obtained over time; identifying one eye in die living human subject as a targeted eye; using said non-invasive automated apparatus on a first measurement occasion to continuously monitor, repetitively measure, and cumulatively record d e pupil diameter size of said targeted eye in die living human subject over die prechosen duration to provide primary information of pupU diameter size for said targeted eye; administering at least one neural transmitter mediator to said targeted eye of die living human subject in an amount insufficient to cause marked constriction of pupU diameter over time in a person not afflicted widi Alzheimer's disease, said neural transmitter mediator being selected from die group consisting of cholinergic agonists and adrenergic antagonists; waiting a predetermined interval of time sufficient for said administered neural transmitter agonist to act upon said targeted eye; then using said non-invasive automated apparatus on at least a second measurement occasion to continuously monitor, repetitively measure, and cumulatively record die pupil diameter size of said targeted eye in die living human subject over die prechosen duration to provide secondary infoπnation of pupil diameter size for said targeted eye after being acted upon by said administered neural transmitter mediator; and deterrnining such change in pupil diameter size for said targeted eye as may have occurred over said time interval by comparing said primary information with at least said secondary information, a marked decrease in pupil diameter size for said targeted eye diagnostically establishing that living human subject as being afflicted widi Alzheimer's disease. 16. The diagnostic metiiod as recited in claim 15 wherein said neural transmitter mediator is an agent selected from the group consisting of pUocarpine hydrochloride, pUocarpine nitrate, and carbachol. 17. The diagnostic metiiod as recited in claim 15 wherein said neural transmitter mediator is an agent selected from the group consisting of physostigmine sulfate and physostigmine salicylate. 18. The diagnostic method as recited in claim 15 wherein said neural transmitter mediator is an agent selected from d e group consisting of demecarium bromide and echothiophate iodide. 19. The diagnostic metiiod as recited in claim 15 wherein said neural transmitter mediator is timolol maleate. 20. A non-invasive metiiod for diagnosing Alzheimer's disease in a living human subject, said diagnostic metiiod comprising die steps of: providing a non-invasive automated apparatus which can continuously monitor pupU diameter size over time, repetitively measure pupU diameter size over time for a prechosen duration ranging from about less than 1 second to about 5 minutes, and cumulatively record such monitored and measured pupU diameter size information as is obtained over time; identifying one eye in die living human subject as a targeted eye; using said non-invasive automated apparatus on a first measurement occasion to continuously monitor, repetitively measure, and cumulatively record die pupU diameter size of said targeted eye in die living human subject over the prechosen duration to provide primary informational data of pupil diameter size for said targeted eye; administering at least one neural transmitter mediator to said targeted eye of d e living human subject in an amount insufficient to cause marked changes in pupU diameter over time in a person not afflicted widi Alzheimer's disease, said neural transmitter mediator being selected from the group consisting of cholinergic agonists, cholinergic antagonists, adrenergic agonists, and adrenergic antagonists; waiting a predetermined interval of time sufficient for said administered neural transmitter mediator to act upon said targeted eye; then using said non-invasive automated apparams on at least a second measurement occasion subsequently to continuously monitor, repetitively measure, and cumulatively record the pupil diameter size of said targeted eye in die living human subject over the prechosen duration to provide at least secondary informational data of pupil diameter size for said targeted eye after being acted upon by said administered neural transmitter mediator; and determining the rate of pupil diameter change for said targeted eye as may have occurred over said time interval by comparing said primary informational data with at least said secondary informational data, a rapid rate of pupU diameter change for said targeted eye diagnostically establishing tiiat living human subject as being afflicted widi Alzheimer's disease. 21. A non-invasive metiiod for diagnosing Alzheimer's disease in a living human subject, said diagnostic metiiod comprising die steps of: providing non-invasive apparatus means for (a) introducing photostimulating visible light of predetermined wavelengtii and intensity to d e eye on-demand sufficient to cause a constriction of die pupU, and (b) determining d e velocity of pupU constriction caused by said introduced photostimulating visible light; identifying one eye in die living human subject as a targeted eye; admi istering at least one neural transmitter mediator to said targeted eye of die living human subject in an amount insufficient to cause a marked change in pupUlary dynamic response in a person not afflicted widi Alzheimer's disease, said neural transmitter mediator being selected from me group consisting of cholinergic antagonists and agonists; waiting a predetermined interval of time for said administered neural transmitter mediator to act upon said targeted eye; tiien introducing photostimulating visible light of predetermined wavelengtii and intensity to the targeted eye sufficient to cause a constriction of die pupil using said non-invasive apparams means; and determining pupil constriction velocity for said photostimulated targeted eye using said non-invasive apparatus means, a marked decrease in pupil constriction velocity for said targeted eye widi respect to a pre-established normative standard diagnostically establishing that living human subject as being afflicted widi Alzheimer's disease. 22. The diagnostic method as recited in claim 21 wherein said neural transmitter mediator is an agent selected from die group consisting of tropicamide, atropine, homotropine hydrobromide, cyclopentolate hydrochloride, and scopolamine.

Inventors: DAFFNER KIRK R, SCINTO LEONARD, DAFFNER, KIRK, R., SCINTO, LEONARD
Applicants: BETH ISRAEL HOSPITAL, DAFFNER KIRK R, SCINTO LEONARD, BETH ISRAEL HOSPITAL ASSOCIATION, DAFFNER, KIRK, R., SCINTO, LEONARD
Application year: 1995
Main classification: A61B3
IPC codes: A61B3, A61B5, A61K49