Method of treatment of traumatic brain injury

A method of treatment of a mammal, including humans, suffering from traumatic brain injury, which comprises administering to the sufferer a therapeutically effective amount of a butyrolactone derivative.

The present invention is directed to a method of treatment of traumatic 
brain injuries. 
It is widely accepted that severe traumatic brain injuries (TBI) initiate a 
cascade of events that lead to dramatic elevation of intracranial pressure 
(ICP) and dysfunction of cerebrovascular regulatory mechanisms essential 
for survival. Indeed, ischemic brain injury is seen universally in those 
patients who die following severe TBI. Intracranial hypertension (IH) 
following traumatic brain injury is associated with direct effects on 
cerebral perfusion which may be responsible for secondary ischemia. The 
contributions of both post-traumatic cerebral edema and alteration in 
cerebral blood volume to ICP appear to vary based on the length of time 
after the primary mechanical insult. This combination of vasomotor 
dysfunction and abnormalities in vascular permeability is characteristic 
of acute inflammation. 
The present invention now provides a method of treatment of traumatic brain 
injuries in mammals, including humans, by administering to a mammal 
suffering from traumatic brain injury a therapeutically effective amount 
of a butyrolactone derivative of the formula 
##STR1## 
wherein: 
R.sub.1 is selected from the group consisting of hydrogen and lower alkyl; 
R.sub.2 is selected from the group consisting of hydrogen, lower alkyl and 
CH.sub.3 YCH.sub.2 --; 
R.sub.5 and R.sub.6 are selected from the group consisting of hydrogen and 
lower alkyl and may be the same or different; 
R.sub.8 is selected from the group consisting of hydrogen and lower alkyl; 
R.sub.7 may be R.sub.8 or 
##STR2## 
R.sub.9 is selected from the group consisting of 
##STR3## 
R.sub.10 and R.sub.11 are selected from the group consisting of hydrogen, 
lower alkyl, phenyl and hydroxyl substituted lower alkyl and may be the 
same or different; 
when R.sub.7 contains a hydroxyl group in the .alpha., .beta. or .gamma. 
position, R.sub.7 may form the hemiketal ring closure at carbon 3 of the 
butyrolactone with protonation of the carbonyl group on the same carbon 
atom; 
R.sub.12 is selected from the group consisting of hydrogen, lower alkyl and 
lower haloalkyl; 
R.sub.13 is lower alkyl; 
m is 2, 3 or 4; 
n is 1, 2 or 3; 
X is O, S or NH; and 
Y is O or S. 
Compounds of formulas (I) to (V) are known and have been proposed for use 
for various therapeutic indications. U.S. Pat. Nos. 4,518,611, 4,620,014, 
4,833,808, 4,883,813, 5,098,933 and 5,102,909, which are incorporated 
herein by reference thereto, describe these compounds, methods for their 
preparation and methods for formulating these compounds into 
pharmaceutical compositions. 
U.S. Pat. No. 4,883,813 proposes the use of certain butyrolactone 
derivatives for treatment of inflammation in mammals, such as acute 
inflammation. However, none of the prior art has proposed the use of 
compounds (I) to (V) for the treatment of traumatic brain injury. As is 
known, the treatment of traumatic brain injury with steroidal or 
non-steroidal anti-inflammatory drugs is not likely to be successful due 
to a number of factors, including the inability of conventional 
anti-inflammatory agents to cross the blood-brain barrier. It was therefor 
unexpected that the butyrolactone derivatives used in the present 
invention would be useful in the treatment of traumatic brain injury. 
It is presently preferred to use the following compounds in the treatment 
of traumatic brain injury according to the present invention: 
##STR4## 
wherein R.sub.12 is lower alkyl, m is 2, 3 or 4, and R.sub.13 is hydrogen, 
lower alkyl or lower haloalkyl, and Y is O or S. 
It is most preferred at present to use the compound referred to as 
Methoxatone, which has the formula 
##STR5## 
It is presently believed that compounds (I) to (V) exert their attenuating 
effect on post-traumatic intracranial hypertension by reducing both the 
cerebrovascular permeability defects that promote edema formation, as well 
as blunting the, as yet, undefined stimulus for inflammatory cell 
infiltration. It is believed that the biochemical foundation for the 
clinical manifestations of TBI, namely cerebrovascular permeability and 
vasomotor dysfunction, lies in the generation of inflammatory mediators by 
resident cells of the central nervous system and/or infiltrating 
leukocytes. Based on the current understanding of the pathophysiology of 
TBI, the anti-inflammatory effects of compounds (I) to (V) would be 
therapeutically beneficial in the treatment of severe head injury. 
It is presently preferred to administer compounds (I) to (V) parenterally, 
such as intravenously, in a bolus, so as to obtain the most rapid delivery 
of the active agent to the brain. A suitable daily dosage for obtaining 
attenuation of the effects of traumatic brain injury is from about 10 to 
about 1000 mg/kg body weight, although the optimum dosage of the compound 
(I) to (V) will be determined by the physician taking into account the 
age, weight and general health of the subject. The daily dosage may also 
be administered in one or several treatments over a period of time, such 
as by way of single or multiple doses per day or from sustained release 
compositions. 
The compounds (I) to (V) may be administered alone or, more usually, in the 
form of a pharmaceutical composition comprising a therapeutically 
effective amount of the active agent in combination with an inert 
pharmaceutically acceptable diluent or carrier therefor. The choice of the 
diluent or carrier will be determined by the route of administration, the 
solubility of the compound and standard pharmaceutical practice. 
Oral and parenteral dosage units will be prepared in accordance with 
standard procedures and may contain the selected active compound (I)-(V) 
as the only or principal active ingredient in the composition. Any of a 
wide variety of known inert excipients may be employed to prepare useful 
compositions. These include, for example, dextrose, starch, talc, various 
types of clay, mineral oil, cottonseed or sesame oil, as well as water or 
various miscible and immiscible aqueous compositions in which the 
therapeutic agent is soluble or may be suspended with the aid of known 
surfactants. 
For buccal and sublingual administration, the active ingredient can be 
formulated in tablet form with watersoluble binding agents, such as 
lactose or other palatable carbohydrates. 
For rectal administration, suppositories or inserts containing the active 
ingredient dispersed in such reagents as cocoa butter, petrolatum, or 
other natural lubricants or in a synthetic emollient such as polyethylene 
glycol 1000 or polyethylene glycol 4000 may be used. 
It may be convenient to administer Compounds (I) to (V) from sustained 
release dosage forms. A number of compositions suitable for such 
preparations are known and can be usefully employed. For oral, sustained 
release administration, the selected therapeutic agent may be in a time 
disintegrating tablet or pellet coated with various thicknesses of known 
materials such as carnauba wax, cellulose esters and ethers, fats, 
keratin, gluten or various natural or synthetic esters. Tablets in which 
the selected agent is contained in a slowly dissolving core such as 
dehydrogenated castor oil or fatty acids can also be employed. 
Alternatively, the active material can be bound to an ion exchange resin 
such as a sulfuric acid type cation exchange resin. 
A number of transdermal formulations are possible for use in the practice 
of this invention. They are discrete dosage forms in construction systems 
which, when applied to the skin, deliver the therapeutic agent through the 
skin at a controlled rate for systemic circulation. A transdermal device 
typically comprises an outer covering barrier, a drug reservoir which may 
have a rate of release controlling membrane, a contact adhesive applied to 
some or parts of the device at the device/skin interface and a protective 
layer which is removed before applying the device. The drug reservoir is 
normally some type of polymer matrix such as a polyvinylpyrrolidone or a 
silicone polymer from which the drug is slowly released. A microporous 
membrane such as a polypropylene film may serve as a membrane to control 
the rate of release. 
The compounds (I)-(V) may also be used in association with other 
therapeutic agents including, for example, antibiotics or antiviral agents 
.

The following Example illustrates the present invention through the use of 
an accepted animal model for traumatic brain injury. This Example refers 
to FIGS. 1-5 of the accompanying drawings, and to Tables I-III, which 
follow the Example. 
EXAMPLE 
Twenty female miniature Yucatan swine (Charles River Laboratories) were 
employed in this study. Experiments were conducted in accordance with the 
Animal Welfare Act and approved by the Institutional Animal Care and Use 
Committee of Cornell University Medical College. The animals were 
premedicated with ketamine and xylazine, weighed, and brought to the 
surgical laboratory where anesthesia was induced with isoflurane by mask. 
They were then intubated, placed supine, and ventilated with an anesthetic 
gas mixture (oxygen 23%, nitrogen 75%, isoflurane 2%) to maintain normal 
arterial blood gases. The left femoral artery and vein were cannulated for 
continuous arterial blood pressure monitoring and placement of a 
flow-directed pulmonary artery catheter for central venous pressure (CVP), 
pulomary artery (PA) and pulmonary capillary wedge pressure (PCWP) 
monitoring. A cystotomy was performed and a 14 French Foley catheter was 
placed for urine output monitoring. The right subclavian vein was 
cannulated for venous access. A rectal thermistor was placed for core 
temperature monitoring. The animals were then repositioned prone and the 
heads were immobilized in a frame. The skull was exposed though a sagittal 
scalp incision and both cerebral ventricles were cannulated using the 
right-angle technique. Intracranial pressure was continuously monitored 
with a fiberoptic pressure transducer (Camino Labs, San Diego, Calif.). An 
18-gauge teflon catheter placed in the contralateral ventricle confirmed 
ICP by manometry. The sagittal sinus was catheterized with a PE-10 
polyethylene cannula for sagittal sinus pressure (SSP) monitoring. A 14-mm 
burr hole was made through the right frontal bone, with care taken to 
avoid injury to the underlying dura. A hollow stainless steel tube (injury 
screw), 14 mm in outer diameter and threaded at one end, was screwed into 
the burr hole. The injury screw was cemented in place to form a 
water-tight seal with methylmethacrylate, and attached to a fluid 
percussion device by a 3-cm length of nondistensible Tygon.RTM. tubing. 
The system was filled with normal saline at 37.degree. C. and purged of 
air. 
Animals were maintained normothermic with a heating lamp, and euvolemic 
with the intravenous infusion of Ringer's lactate to maintain CVP between 
2 and 5 mm Hg and urine output .gtoreq.0.5 ml/kg/hour during the baseline 
period. Animals were allowed to equilibrate for a 30-minute period prior 
to experimental injury. The following physiological variables were 
continuously monitored using an analog-to-digital conversion data 
acquisition system: mean arterial blood pressure (mABP), CVP, PA, PCWP, 
SSP, ICP, heart rate (HR), inspired (FiO.sub.2) and expired (FeO.sub.2) 
oxygen concentrations, expired isoflurane concentration, end tidal 
CO.sub.2 (PetCO.sub.2), and core temperature. Arterial blood gas 
determinations were made at 30 minute intervals in 300 .mu.l samples 
(Blood gas analyzer Model 288, Ciba Corning, Medfield, Mass.). 
Determination of Brain Compliance 
Brain compliance was determined using the single injection method decribed 
by Marmarou, et al. For each of the determinations, made at least 5 
minutes apart, a 0.5 mL bolus infusion of normal saline, maintained at 
37.degree. C., was given over 0.5 seconds. The ICP immediately prior to 
injection was defined as P.sub.o, and the highest pressure recorded after 
injection was defined as P.sub.max. The pressure-volume index (PVI) was 
calculated using the following equation: 
EQU PVI=V/Log P.sub.o /P.sub.max 
where V=volume of the bolus infused. Once the PVI was calculated, brain 
compliance was derived from the equation: 
EQU Compliance=0.4343 PVI/P 
where P is the ICP at the point when the compliance measurement was 
determined and 0.4343 is a constant. A minimum of three determinations 
were made during the baseline period in all animals. 
Fluid Percussion Injury 
Experimental traumatic brain injury was induced by fluid percussion injury, 
modified from the method described by Sullivan. A 2.5 Atm barotraumatic 
injury was delivered to the brain over 20 to 25 milliseconds through the 
right frontal injury screw. Pressure was measured at the distal end of the 
fluid percussion piston by a high resolution transducer (Statham, Puerto 
Rey, Puerto Rico) Following percussion, the injury screw was disconnected 
from the fluid percussion device, the screw removed from the skull, and 
the cranial detect covered with bone wax to maintain the injured area as 
close to core temperature as possible. Previous work has demonstrated that 
this degree of barotrauma resulted in a reproducible injury characterized 
by histopathological evidence of a severe frontal lobe injury, including 
development of frontal contusion and intracranial hypertension, without 
brain stem deformation that could affect cardiopulmonary performance. 
Animals were divided into three groups. Group I (n=6) was subjected to TBI, 
Group II (n=6) was subjected to TBI and received an i.v. bolus injection 
of methoxatone ("METH") 100 mg/kg, one hour following injury, Group III 
(n=8) was a surgical sham. 
Preparation of Drug 
Crystalline, lyophilized methoxatone was maintained at -20 C until it was 
reconstituted in phosphate buffered saline to a concentration of 100 mg/ml 
immediately prior to each experiment. Methoxatone at a dose of 100 mg/kg 
body weight was delivered via intravenous bolus injection 60 minutes 
following injury. Serum samples were obtained at various time points 
following administration of the compound for the determination of serum 
concentration by High Pressure Liquid Chromatography (HPLC). 
Determination of Cerebral Blood Volume 
Since cerebral blood flow and volume increase linearly with PaCO2 (Hampton 
et al), the rate of respiration was manipulated to produce varying 
PaCO.sub.2 (PetCO.sub.2) in order to determine the changes in CBV as 
measured by cerebral cortical reflectance photoplethysmography and 
Technetium-99 m labeled red blood cells 
Cerebral Cortical Reflectance Photoplethysmography 
A 14 mm burr hole was made in the frontal region, contralateral to the 
injury screw, in all animals for epidural placement of a flexible 
reflectance photoplethysmography probe. The probe consisted of miniature 
red and infrared light-emitting diodes (LED's) and a silicon 
photodetecting diode mounted on a flexible circuit board. Output from the 
photodetector was selectively tuned to provide data on photostimulation by 
specific wavelengths. The probe was connected to a photodemodulation 
circuit and an analog-to-digital converter connected to a microcomputer 
(Macintosh II, Apple Computer, Cupertino, Calif.), Reflected red and 
infrared photoplethysmograms were employed to evaluate cerebral cortical 
blood volume and oxygen saturation of hemoglobin (SaO.sub.2), Amplitudes 
of the reflected signals were used in the calculation of SaO.sub.2 
(oximetry) and as an index of cerebral blood volume. 
Hemoglobin within cerebral cortex, illuminated by the LED's on the surface 
of the probe, reflects red and infrared light which is detected by the 
photodiode. The intensity of the red and infrared signals varies with the 
cardiac cycle and is dependent upon the SaO.sub.2. The oximetry technique 
analyzes the pulsatile (referred to as the a.c. component), rather than 
absolute, non-pulsatile (referred to as the d.c. component), reflected 
light intensity of red and infrared photoplethysmograms, measured at 660 
and 910 nm, respectively. The wavelengths chosen represent portions of the 
spectral region where the absorption coefficients of reduced and 
oxygenated hemoglobin in tissue are markedly different (660 nm), and where 
they are relatively similar (910 nm). An isobestic wavelength (820 nm), 
where the absorption coefficients for both oxygenated and reduced 
hemoglobin are roughly the same, was used as a reference for calibration. 
When the reflected d.c. signal remains constant, the amplitude of the 
reflected a.c. signal varies as a function of total hemoglobin within the 
illuminated tissue using algorithms previously employed in near-infrared 
spectrophotometry by Wyatt, et al. The sensitivity of this system to 
changes in cerebral perfusion in response to hypo- and hypercarbia was 
previously documented. 
Prior to each experiment, correlation of calculated cerebral cortical 
arterial hemoglobin saturation to SaO.sub.2 was established. 
Technetium-99m(.sup.99m Tc) Labeled Red Blood Cells 
Cerebral blood volume (CBV) changes were simultaneously measured in some 
animals using radioactively labeled red blood cells (RBCs) to calibrate 
the reflectance photoplethysmographic technique. Briefly, 10 mL of whole 
blood was labeled with .sup.99m Tc using the modified in vivo method of 
Callahan, et al. This technique assures rapid (&gt;90% efficiency within 5 
minutes) and reproducible labeling. The labeled RBC's were then reinfused 
and regional CBV was measured directly by a gamma collimator placed over 
the site of the reflectance photoplethysmographic probe (FIG. 1). The 
collimator employed a 20 percent window over the 140-keV photopeak of 
.sup.99m Tc. Arterial blood samples (2.0 mL), obtained at the time of 
gamma emmission data collection, were divided into three equal aliquots. 
One aliquot was used for the determination of total hemoglobin by 
co-oximetry (blood gas analyzer Model 288, Ciba Corning, Medfield, Mass.); 
the hematocrit was determined by microcentrifugation. The two remaining 
aliquots were placed in a well counter and total blood sample counts were 
decay corrected to the time they were drawn by the equation: 
EQU C.sub.o =Ce.sup..lambda.t 
where C.sub.o =decay-corrected counts, Ce=decayed counts, 
.lambda.=0.693/6.02 hr physical half life of .sup.99m Tc, and t=the time 
between when the sample was drawn and when it was counted. These data were 
then employed to calculate CBV, expressed as mL/100 g tissue, from the 
total counts obtained from the collimators. 
Histological Analysis of Cerebral Cortex 
Immediately following sacrifice, the supratentorial brain was removed 
intact and representative sections from the right frontal lobe directly 
under the injury screw, the left frontal lobe, the left occipital lobe and 
left parietal lobe were sharply dissected free and placed in neutral 
buffered formalin. The tissue was fixed for a minimum of 96 hours and then 
processed for light microscopy. Sections were then evaluated under blinded 
conditions, and graded according to the following convention: 
Cerebrovascular Injury Index 
Grade 0 Normal brain, no evidence of cerebrovascular inflammation, no 
intraparenchymal PMN's, no evidence of PMN margination or diapedesis 
Grade 1 PMN margination, minimal extravasation of RBC's, normal endothelium 
Grade 2 Extensive PMN margination with evidence of diapedesis of PMN's, 
with normal or swollen endothelium 
Grade 3 Extensive PMN Margination with diapedesis and intraparenchymal 
PMN's.+-.extravastion of RBC's, swollen endothelium 
Grade 4 Extensive PMN Margination with diapedesis and intraparenchymal 
PMN's.+-.extravastion of RBC's, with evidence of PMN phagocytosis of 
neurons and/or glia, gross disruption of endothelium and/or vascular 
basement membrane 
Brain Specific Gravity Determination 
Representative sections of brain tissue from identical regions employed for 
histological analysis were immediately placed in sealable polyethylene 
pouches and kept on ice until delivered to the laboratory for 
determination of tissue specific gravity. Three 1 mm cubes of tissue were 
sharply dissected from each specimen and placed in a kerosene/bromobenzene 
density gradient column and allowed to equilibrate. The column was 
calibrated against beads of known density. Measurements were taken after 
2, 3, 5 and 10 minutes on the column to factor out non-specific 
evaporation of surface water in different specimens. 
Data Analysis 
Physiological data are expressed as mean.+-.standard error of the mean. 
Differences between groups at each time point were compared using the 
Wilcoxon nonparametric analysis. A two-way analysis of variance (ANOVA) 
was employed to examine differences in individual variables within groups 
with respect to time. Correlation of individual physiological variables 
between multiple groups was evaluated using the Spearman ranked 
correlation coefficient. Histological data was compared using a two-sample 
t-test assuming unequal variances. A significance level of p&lt;0.05 was used 
throughout this study. 
RESULTS 
Calibration of Photoplethysmographic Method of Determining CBV 
To evaluate the accuracy of the photoplethysmographic technique for 
determing CBV changes in real time, simultaneous determinations were made 
by counting radioactively labeled RBC's in the same tissue region. Both 
.sup.99m Tc decay from labeled RBC's and the red a.c. amplitude derived 
from reflectance photoplethysmography were well correlated with changes in 
CBV that occured in response to variations in PET CO.sub.2 (FIG. 1a). This 
relationship was significant (p=0.00001) throughout the range of 
photodetector sensitivity of the system (FIG. 1b), and when counts were 
corrected for decay. Total counts were then converted to a blood volume 
measurement based on the results of direct counting of whole blood 
specimens drawn during the experiment. From this relationship, calculation 
of CBV was made from the red a.c. amplitude alone. 
Physiologic data, obtained 30 minutes following surgical preparation, 
demonstrated no statistically significant differences in baseline ICP, 
mABP, CVP, PCWP, and SVR between groups, Arterial blood gases and 
temperature were unchanged from baseline values throughout the duration of 
the experiment in all groups. Animals subjected to fluid percussion injury 
developed a significant, transient systemic hypertension following TBI 
that lasted approximately 30 minutes and then returned to baseline values 
(FIG. 2). Arterial blood pressure slowly decreased during the 6 hour 
experimental period in all animals, however, these changes in mABP were 
not statistically different when values were compared in head injured and 
sham treated animals. 
Dramatic intracranial hypertension occurred immediately following TBI in 
injured animals (FIG. 3), rising to significantly higher levels from 
baseline (Group I; 22.+-.3 vs 8.+-.3, p&lt;0.001 and Group II; 21.+-.3 vs 
9.+-.2, p&lt;0.001) within minutes of injury, and returning to near baseline 
levels within 60 minutes. ICP continuously rose in Group I during the 
subsequent 5 hours, and at 6 hours was significantly higher than baseline 
values (27.+-.3 vs 8.+-.3, p&lt;0.001) as well as compared to sham (27.+-.3 
vs 7.+-.2, p&lt;0.001). In contrast, animals treated with Methoxatone showed 
a significant attenuation of this increase in ICP especially between 210 
and 360 minutes post injury (FIG. 3), and at 6 hours was significantly 
lower than Group I (14.+-.3 vs 27.+-.3, p&lt;0.05) In addition, the immediate 
post-injury rise in ICP that occurred during the period of systemic 
arterial hypertension was accompanied by an increase in CBV to more than 
double the baseline values in Groups I and II (19.2.+-.1.4 vs 8.9.+-.1.1 
mL/100 g tissue, p&lt;0.05) at the time of the greatest rise in ICP. Although 
CBV decreased from this peak at the time of injury, it remained 
significantly elevated above baseline levels throughout the experimental 
period in Group I. CBV returned to near baseline values in Group II by 60 
minutes and remained at this level throughout the experimental period. 
Arterio-venous oxygen content differences across the brain significantly 
decreased (1.9.+-.0.1 vs 3.8.+-.0.3 mL/dL, p&lt;0.05) during the immediate 
post-traumatic rise in ICP and CBV, and was inversely related to the 
photoplethysmographically calculated cerebral cortical SaO.sub.2. Oxygen 
extraction then gradually increased during the post-traumatic period and 
was significantly higher than baseline values from 3 to 6 hours following 
injury in Group I. In addition, calculated cerebral cortical SaO.sub.2 
initially rose after injury and then gradually declined to significantly 
lower than baseline values after 4 hours. Oxygen extraction was closer to 
baseline values in the 3 to 6 hour post-injury period in Group II. 
Brain water content determined by gravimetry was significantly higher in 
both the directly injured right frontal lobe and the adjacent left frontal 
lobe of animals in Group I compared to Group III controls (FIG. 4, Table 
1). (Lower specific gravities correlate well with increased brain edema 
due to injury.) However, tissue water content of the injured right frontal 
lobe of animals treated with the Methoxatone was lower than untreated 
animals and approached statistical significance (p=0.061). The brain water 
content of the adjacent frontal lobe of treated animals was not 
statistically different from that present in the sham controls. 
Histological analysis of the right and left frontal lobes of untreated, 
injured animals demonstrated evidence of cerebrovascular injury and 
inflammatory cell infiltration according to the grading scale described 
above. Based on the cerebrovascular injury index (CVII), the injured right 
and left frontal lobes in untreated animals showed significantly greater 
evidence of injury and inflammation than in the control group (Group III) 
(Table 2, FIG. 5 A-I). In contrast, the right frontal lobe in Methoxatone 
treated animals showed significantly less evidence of injury 
(CVII=1.7.+-.0.5 vs 3.2.+-.0.6, p&lt;0.05), while the left frontal lobe in 
Group II was indistinguishable from uninjured controls. 
SUMMARY 
The pathophysiologic events that follow experimental TBI are well 
described. The development of intracranial hypertension, alterations in 
CBV and increase in brain tissue water content, increased oxygen 
extraction and histologic evidence of cerebrovascular injury and 
inflammation are characteristic of acute severe mechanical brain injury in 
this model. Methoxatone significantly attenuated the intracranial 
hypertension seen in untreated animals following TBI. Methoxatone appeared 
to exert its effect within 60 minutes of administration. As seen in FIG. 
3, ICP remained significantly lower in the Methoxatone treated group 
compared to untreated controls, from 150 minutes following administration 
to the end of the experimental period. Although ICP was still 
significantly elevated in treated animals compared to sham controls, 
Methoxatone appears to protect the injured brain from the uncoupling of 
metabolic demand and cerebral blood flow as evidenced by the preservation 
of near-baseline oxygen extraction, compared to significantly elevated 
oxygen extraction in untreated animals. 
Methoxatone treated animals developed less tissue edemas as measured by 
tissue specific gravity, approaching statistical significance (p=0.061) 
compared to untreated injured animals. This data suggests that Methoxatone 
reduces the post-traumatic defect in cerebrovascular permeability which 
promotes the movement of water into the tissue contributing to cerebral 
edema. 
Methoxatone had a significant effect on post-traumatic inflammation, as 
evidenced by the lower CVII Histopathology scores in treated animals 
compared to untreated controls. Morphological evidence of cerebrovascular 
injury, namely infiltration of inflammatory cells, neutrophil margination 
and diapedesis, and neutrophil phagocytosis of pyknotic neurons was 
markedly reduced in treated animals. 
TABLE 1 
__________________________________________________________________________ 
Group I Group II Group III 
Region TBI TBI + METH 
Sham 
__________________________________________________________________________ 
Right Frontal (Injury) 
1.033828 .+-. 0.00114 
1.036264 .+-. 0.00122 
1.040233 .+-. 0.00080 
Left Frontal (adjacent) 
1.035506 .+-. 0.00089 
1.037278 .+-. 0.00089 
1.039088 .+-. 0.00096 
__________________________________________________________________________ 
TABLE 2 
______________________________________ 
Results: Cerebrovascular Injury Index 
R Frontal L Frontal 
Group (inj) (adjacent) 
______________________________________ 
TBI 3.2 .+-. 0.6 
1.3 .+-. 0.7 
TBI + Meth 1.7 .+-. 0.5* 
0.5 .+-. 0.3** 
Sham 0.5 .+-. 0.2 
0.6 .+-. 0.2 
______________________________________ 
*p &lt; 0.05 
**p &lt; 0.01 
TABLE 3 
__________________________________________________________________________ 
Photomicrographs A-I of FIG. 5 
__________________________________________________________________________ 
A-(63 .times.) Right frontal lobe section, sub-meningeal cortex, taken 6 
hours 
following intracerebroventricular infusion of 20 uM LTC4. An intense 
meningovasculitis, characterized by margination and diapedesis of 
neutrophils, infiltration of neutrophils into the parenchyma, and 
meningitis 
with a dense neutrophil-rich exudate in the subarachnoid space. 
B-(250 .times.) Higher magnification from the section in A. 
Intraparenchymal 
neutrophils are shown phagocytizing an astrocyte. Multiple neutrophils 
are 
seen throughout the neuropil. 
C-(160 .times.) Right frontal lobe section, sub-meningeal cortex, taken 6 
hours 
following intracerebroventricular infusion of 20 uM LTC4. Free 
intraparenchymal neutrophils (.fwdarw.A) Phagocytosis of pyknotic neurons 
by 
intraparenchymal neutrophils (.fwdarw.B) 
D-(160 .times.) Right frontal lobe section, sub-meningeal cortex, taken 6 
hours 
following experimental TBI. Neutrophils are seen diapedesing through an 
apparently intact parenchymal vessel. Note the contraction artifact halo 
around the vessel indicating pre-fixation perivascular edema. 
E-(160 .times.) Right frontal lobe cortical section, taken 6 hours 
following 
experimental TBI. Free intraparenchymal neutrophils seen (.fwdarw.A). 
Neutrophil with intracellular inclusions representing phagocytized 
debris 
(.fwdarw.B). 
F-(250 .times.) Right frontal lobe cortical section, taken 6 hours 
following 
experimental TBI in animals treated with METH. Intraparenchymal 
arteriole 
without evidence of neutrophil margination. Numerous pyknotic cells 
without intraparenchymal neutrophil infiltration. 
G-(63 .times.) Right frontal lobe cortical 
section, taken 6 hours following 
experimental TBI in animals treated with 
METH. Intraparenchymal vessel ( ) with 
small contraction artifact halo. Pyknotic 
neuron (&gt;) and only isolated 
intraparenchymal neutrophil (.fwdarw.)--. 
H-(160 .times.) Right frontal lobe cortical section, taken 6 hours 
following 
experimental TBI in animals treated with METH. Numerous pyknotic 
neurons with very few intraparenchymal neutrophils. 
I-(63 .times.) Right frontal lobe cortical section from sham control. 
Normal 
cellularity, no pyknotic cells, no intraparenchymal neutrophils. 
__________________________________________________________________________