PEA-15 is a phosphoprotein that binds and regulates ERK MAP kinase and RSK2 and is highly expressed throughout the brain. PEA-15 alters c-Fos and CREB-mediated transcription as a result of these interactions. To determine if PEA-15 contributes to the function of the nervous system we tested mice lacking PEA-15 in a series of experiments designed to measure learning, sensory/motor function, and stress reactivity.We report that PEA-15 null mice exhibited impaired learning in three distinct spatial tasks, while they exhibited normal fear conditioning, passive avoidance, egocentric navigation, and odor discrimination. PEA-15 null mice also had deficient forepaw strength and in limited instances, heightened stress reactivity and/or anxiety. However, these non-cognitive variables did not appear to account for the observed spatial learning impairments. The null mice maintained normal weight, pain sensitivity, and coordination when compared to wild type controls.We found that PEA-15 null mice have spatial learning disabilities that are similar to those of mice where ERK or RSK2 function is impaired. We suggest PEA-15 may be an essential regulator of ERK-dependent spatial learning. PEA-15 is a 15 KDa phosphoprotein that regulates both ERK MAP kinase and death receptor apoptosis pathways . PEA-15 The ERK MAP kinase pathway is involved in learning and memory. In particular, it is implicated in spatial learning and fear conditioning . Both ofBecause PEA-15 can regulate both ERK and RSK2 we assessed PEA-15 knockout (KO) mice in a series of tests that measure learning, sensory/motor function, and stress reactivity. We found that deletion of PEA-15 protein in C57BL/6J mice causes specific defects in spatial learning abilities.PEA-15 is expressed at high levels throughout the nervous system. PEA-15 null (KO) mice have been reported to have normal brain sizes and astrocyte numbers and to be born in roughly normal ratios . No diff = 4.27; p < 0.05]  = 10.79; p < 0.05]  = 6.25; p < 0.05]. There was also a trend for KOs to make a first open arm entry more quickly than WTs. Therefore KOs tended to exhibit higher levels of anxiety or fear with a concomitant increase in exploration of the maze.In elevated plus maze tests KOs (n = 13) deposited more bolli in the maze than WTs (n = 9), [F] Figure  suggesti] Figure . This di] Figure . HoweverAn alternative test of anxiety and exploration is the Open Field test. In this test KO and WT mice exhibited similar numbers of total movements in the open field Figure . However = 5.34; p < 0.05] and spent significantly less time in the white well lit side  (data not shown). This difference was not due to differences in activity between the groups, since both made a similar number of crossings between the two sides of the apparatus. This test is further evidence that KO mice suffer increased levels of anxiety or fear.In the Black/White Preference test, mice have a propensity to spend more time in the black, dimly lit half of the preference chamber presumably because they find this chamber less stressful than the well-lit white half and because animals are introduced into the apparatus on the black side. In this test, both KO and WT groups spent more of the total time during testing in the black/dim side of the chamber as is normal. However, KO mice made their initial crossing into the white side of the chamber more slowly than WT mice . Groups did not differ in their latency to explore the escape straight alley, nor did they show a preference for either end of the apparatus (start box or open end). Hence, KO mice do not appear to suffer increased fear as measured in this test. In total, the differences noted above for the elevated plus maze and the black/white preference test suggest a pattern of behavior in KO mice  that does not reflect a consistent pattern of differences in stress or anxiety related behaviors. Thus these differences may reflect other, unidentified causes.As a separate measure of fear we examined KO mouse performance in the Straight alley/Escape test. The startle stimulus (bright light combined with increased air flow) used during this test typically elicits rapid running (an "escape" response), which serves as an index of fear. Though the escape distance for PEA-15 null mice was only 63% of that for wildtype mice  = 18.27; p < 0.001]. This difference was not due to less running behavior during the light cycle, when all mice spent significantly less time running, but was due to KOs spending significantly less time running during the night cycle   = 3.49; p < 0.07]  mice to react slower to the painful stimulus than WT mice (n = 9) [F] Figure . Differe = 4.69; p < 0.05]  = 20.87; p < 0.001]  = 8.26; p < 0.01]  = 0.12; n.s.]  = 5.12; p < 0.01]. However, KOs (n = 13) were impaired in their mean latency to locate the reinforcer  on the first training trial (before any learning could occur)  = 4.21; p < 0.05] and committed more 'errors'  than WTs.In odor discrimination, KO mice did not differ from WTs during learning Figure . Both tycur) Fig . KOs alscur) Fig , althougcur) Fig  and 3D.  = 10.95; p < 0.01], but there was no difference between KOs and WTs in performance in this test . To control for individual basal levels of activity and exploration, step-down latencies were calculated by utilization of a ratio of post training latencies as a function of pre-training latencies. Both KO (n = 9) and WT (n = 9) mice significantly increased their latencies to step down after training . This difference was not due to reduced strength or activity of the KO mice since all mice had similar swim speeds  on the first training trial before any learning had occurred  = 3.44; p < 0.0001]. Not surprisingly, WT controls also out-performed KOs during a probe trial after training . KOs spent equivalent search time in the target quadrant as in the other three quadrants  = 4.81; p < 0.05] during the final four trials as measured by latency to navigate the maze and make an arm choice  = 2.03; ns]  mice were impaired compared to WTs (n = 9), [Fe Figure . While a] Figure . Since b = 2.03; p < 0.05], but at no point during training did the KOs differ from the WTs in errors committed . Again, however, the KO mice took significantly longer navigating the maze to make correct arm choices . Again, these results suggest that KO animals require a longer time to execute ultimately accurate decisions under the demands of spatial navigation.The PEA-15 null mice appeared to be impaired in tasks that can be solved using a spatial strategy . To further explore this observation, mice were tested in a modified version of the spatial win/stay task. This test was executed in the same manner as the earlier spatial win/stay task with the exception that animals could correct wrong choices. In the modified win/stay task, mice received an additional day of training . Under these conditions, all mice improved performance during training [FPEA-15 is a small phosphoprotein that binds to ERK and regulates ERK signaling . In partPEA-15 binds tightly to ERK and RSK2 and thereby regulates the outcome of ERK signaling . More reMutations in RSK2 cause the X-linked Coffin-Lowry syndrome . Aside fRecently, morphometric analysis by MRI of Coffin-Lowry patients showed reduced cerebellum and hippocampus volumes . HoweverIn most respects the learning disabilities of PEA-15 null mice are similar to those of mice with impaired ERK activity. However, we found no significant change in fear conditioning in the KO mice, whereas there is literature reflecting a role for ERK in this process ,32. It mWe report that PEA-15 deficient mice exhibit an impaired ability at spatial learning tasks. They also have weaker forepaws than their WT counterparts do. It is therefore possible that the reduced forepaw strength may contribute to the performance of the mice in the spatial learning tests. However, in the spatial water maze, swim speed and path length analysis suggest that the reduced forearm strength in KO mice was not responsible for their impaired performance. The fact that KO mice did not display impaired basal activity in sensory motor tests also supports the premise that the learning impairments in spatial tasks were not contingent on gross differences in sensory/motor function between PEA-15 null and WT mice. In other learning and sensory/motor tests, the PEA-15 null mice showed no deficiencies. Thus the spatial learning defect is most likely independent of the differences in forepaw strength.Our analysis was performed on adult mice in which PEA-15 had been completely deleted. The defects we observe could therefore be due to changes arising from lack of PEA-15 function in early brain development, lack of PEA-15 metabolic function in the behaving adult, or both. The current study cannot distinguish between these possibilities. However, this work provides the framework for additional studies that could address this issue. For example, we could create conditional knock-outs of PEA-15 in the brain in which PEA-15 is deactivated in the adult brain only after development is complete. These mice could then be run through a similar battery of tests to determine whether the effects are due to effects on brain development or adult brain function. These experiments are currently being planned.The ability to learn is a critical trait of many organisms  and the PEA-15 null mice KO; n = 13) and their wild-type controls  were obtained courtesy of H. Chneiweiss (College de France) and were generated by D. Kitsberg and P. Leder by homologous recombination as described [ and theiAnimals were singly housed in clear boxes with floors lined with wood shavings in a humidity- and temperature-controlled vivarium adjacent to testing rooms. A 12 hr/12 hr light/dark cycle was maintained. All behavioral training took place during the middle seven hours of the light cycle. Mice were handled in accordance with National Institutes of Health guidelines for the care and use of animals. All experiments were IACUC approved by the Institutional Review Board of Rutgers, The State University of New Jersey.ad lib food was removed from the animals' home cages at the end of the light cycle approximately 40 hours prior to the start of training (and thus encompassing the "rest" day between successive tasks). During the deprivation period, animals were provided with food in their home cages for 60 min/day during the last 2 hrs of the light cycle, and thus were approximately 16 hrs food-deprived at the time of training or testing. Between each successive test (of learned and unlearned behaviors), animals received a day of rest. Different experimenters (n = 3) trained or tested animals in different tasks, and no experimenter was aware of animals' performance on other tasks, or genotype, until after the completion of the entire battery of tests. In total, animals were assessed in 11 tests of unlearned behavior and fitness, and seven tests of learning. In many of these tasks, multiple measures of performance were obtained such that a total of 12 measures of learned behavior and 25 measures of unlearned performance are reported.All experiments were done blind to the genotype. For the learning tasks that required food deprivation, All animals were tested on 11 tasks that provided 25 measures of unlearned behaviors and/or fitness. A description of these tests and their implementation has been previously reported ,35.1. Body Weight and Food Consumption under Mild Deprivation. The body weights during periods of free feeding were collected every week throughout this series of tests, and the percent change in body weight was recorded after 24 hrs of food deprivation (prior to testing in the Lashley Maze).2. Running Wheel. Animals were housed for 48 hours in cages containing running wheels in the home colony room. Animals were introduced to the running cages at the start of the light cycle on Day 1, and wheel cycles were recorded for 3 hrs as an index of running during initial adaptation. Total cycles on Days 2 and 3 and the percentage of cycles during the light and dark periods were counted.3. Elevated Plus Maze. The maze was constructed of black Plexiglas in the form of a "plus". Each arm of the maze is 6 cm wide, and the maze is suspended 30 cm above a black surface. 8 cm high, black Plexiglas walls surrounded two opposing arms of the maze, and two of the arms were open. The maze was located in a 300 Lux environment. Animals were placed in the center of the maze facing an open arm, and their behavior in the maze was recorded in 1 min blocks for 4 min.4. Open Field Exploration. A square field (46 × 46 cm) with 13 cm high walls of white Plexiglas was utilized. The apparatus was located in a brightly lit room (400 Lux) with a background noise of 65 dB. The field was conceptually divided into a grid comprised of 6 × 6 7.65 cm quadrants, where 20 of the quadrants abutted the outer walls of the field , and 16 quadrants were displaced from the walls and comprised the interior (i.e. "open" quadrants) of the field. Animals were placed in the center of the field. After 20 sec had elapsed , the animals' behavior was monitored for 4-min. Throughout this time the animal's entries into wall and open quadrants were recorded. An entry was recorded whenever both front paws crossed the border of a quadrant.5. Straight Alley Exploration/Magnitude of Escape Response. A straight alley was used that was 30 cm above ground. The alley was 244 cm long and 7 cm wide with 3 cm high walls. The initial 29 cm of the alley was enclosed in 12 cm high walls and an orange acetate ceiling. This portion was designated as the "start box" and the exit from this box could be blocked with a sliding guillotine door made of clear Plexiglas. The interior of the start box was 4 Lux, and the alley beyond the start box was 20 Lux. A startle stimulus could be delivered in the start box. This stimulus was the compound of a bright light (400 Lux) and a high-speed (3000 RPM) fan positioned so that its airflow was directed across the animal and down the alley. The fan raised background noise 50 dB. Animals were placed in the start box with the exit blocked. After 60 sec, the door was raised and animals were allowed to explore the alley for 4 min. The latency for each animal to cross a point in the alley 213 cm from the exit of the start box was recorded as were crossings across the midline of the alley. Both served as an index of exploratory behavior. After 4 min, the animals were returned to the start box where they were again confined for 60 sec. Subsequently the door was raised. At the moment that each animal moved to within 2 cm of the exit and faced the open alley, the compound startle stimulus was initiated and presented for 800 msec. This stimulus typically elicited rapid running (an "escape" response). The distance that the animal ran prior to making a complete stop of forward movement for at least 500 msec was recorded.6. Pain Sensitivity. Upon being placed on a 52.6°C aluminum plate, animals' latency to raise a hind paw and to either lick or shake the paw served as the index of pain sensitivity.7. Balance Beam. Animals were placed on a 40 × 0.7 × 2 cm (l × w × h) beam suspended 30 cm above the ground. In a 4 min test, mice exhibited wide variability in the amount of movement along its length.8. Roto-Rod Suspension. Animals were hung by their front paws from a slowly rotating (8 RPM) rod  suspended 30 cm above ground. Latency to drop from the rod (an index of grip strength) was recorded.9. Balance platform. All four paws of animals were placed on a 3 cm round platform (60 Lux illumination) 30 cm above the ground. Latency to fall (240 sec maximum test duration) from the platform was recorded.10. Screen Climbing and Hanging. Animals were placed on the underside of a wire mesh screen tilted 45° from vertical and suspended 24 cm from ground. The distance moved prior to dropping from the screen  and the latency to drop from the screen was computed.11. Black/White Preference. A 10 × 36 cm chamber divided along its length in two equal halves was utilized. One half was white and brightly lit (100 Lux), and the other half was black and dim (5 Lux). A center wall with a 3 cm square opening that joins the black and white sides divided the two halves. Animals were placed in the black side of the chamber and allowed to explore for 4 min. The latency to first entry into the white chamber, percent of total time in the white chamber, and number of crossings between the black and white chambers was recorded.All animals were tested on seven learning tests, three of which could be solved most efficiently using a spatial strategy. The remaining four tasks are widely asserted to represent different, distinct learning domains, as summarized in Table In a procedure based on one designed by Sara  for ratsA black Plexiglas 60 cm square field with 30 cm high walls was located in a dimly lit (40 Lux) testing room with a high ventilation rate (3 min volume exchange). Three 4 × 4 × 2.0 cm  aluminum food cups were placed in three corners of the field. A food reinforcer (30 mg portions of chocolate flavored puffed rice) was placed in a 1.6 cm deep, 1 cm diameter depression in the center of each cup. The food in two of the cups was covered (1.0 cm below the surface of the cup) with a wire mesh so that it was not accessible to the animal, while in the third cup (the "target" cup), the food could be retrieved and consumed.A cotton-tipped laboratory swab, located between the center and rear corner of each cup, extended vertically 3 cm from the cups' surface. Immediately prior to each trial, fresh swabs were loaded with 25 ul of lemon, almond, or mint odorants (McCormick flavor extracts). The mint odor was always associated with the target food cup. In pilot studies, the odor associated with food was counterbalanced across animals, and no discernible differences in performance was detected in response to the different odors.The Lashley III maze consisted of a start box, four interconnected alleys, and a goal box containing a food reward. Over trials, the latency of rats to locate the goal box decreases, as do their errors . Here, the Lashley III maze was scaled for mice, and parameters were developed that supported rapid acquisition. The maze was constructed of black Plexiglas. A 2 cm wide × 0.1 cm deep white cup was located in the rear portion of the goal box, and 1/2 of a 45 mg BioServe (rodent grain) pellet served as reinforcers. Illumination was 80 Lux at the floor of the maze. The maze was isolated behind a shield of white Plexiglas to prevent extra-maze landmark cues.Food-deprived animals were acclimated and trained on two successive days. On the day prior to acclimation, all animals were provided with three food pellets in their home cages to familiarize them with the novel reinforcer. On the acclimation day, each mouse was placed in the four alleys of the maze, but the openings between the alleys were blocked so that the animals could not navigate the maze. Each animal was confined to the start and subsequent two alleys for 4 min, and for 6 min in the last  alley, where three food pellets were present in the food cup. On the training day, each animal was placed in the start box and allowed to traverse the maze until it reached the goal box and consumed the single food pellet present in the cup. Upon consuming the food, the animal was returned to its home cage for a 20 min interval (ITI), after which it was returned to the start box to begin the next trial. The apparatus was cleaned during each ITI, and the sequence was repeated for five trials. Both the latency and errors  to enter the goal box were recorded on each trial.In order not to duplicate stimuli  used to support associative learning (fear conditioning), we used a variant of the step-down avoidance task that does not rely on shock to motivate behavior. Upon stepping off the platform, animals were exposed to a compound of bright light and loud oscillating noise. A chamber illuminated by dim (< 5 Lux) red light was used for training and testing. Animals were confined to circular ("safe") chamber . The walls and floor of this chamber were white, and the ceiling was translucent orange. The floor was comprised of plastic rods (2 mm diameter) arranged to form a pattern of 1 cm square grids. A clear exit door (3 CM square) was flush with the floor of the safe compartment, and the door could slide horizontally to open or close the compartment. The bottom of the exit door was located 4 cm above the floor of a second circular chamber . This "unsafe" chamber had a clear ceiling and a floor comprised of 4 mm wide aluminum planks that formed a pattern of 1.5 cm square grids that were oriented at a 45° angle relative to the grids in the safe compartment. When an animal stepped from the safe compartment through the exit door onto the floor of the unsafe compartment, the compound aversive stimulus comprised of a bright (550 Lux) white light and oscillating ("siren") noise blast  was initiated.Animals were placed on the platform behind the exit blocked by the Plexiglas door. After 5 min of confinement, the door was retracted and the latency of the animal to leave the platform and make contact with the grid floor was recorded. Prior to training, step-down latencies typically range from 8-20 sec. Upon contact with the floor, the door to the platform was opened and the aversive stimulus (light+noise) was presented for 4 sec, at which time the platform door was opened to allow animals to return to the platform, where they were again confined for 5 min. At the end of this interval animals were returned to their home cages for a 1 hour retention period. Subsequently, mice were returned to the safe platform for 5 min, then the door was opened and the latency of the animal to exit the platform and step onto the grid floor (with no aversive stimulation) was recorded, completing training and testing. The ratio of post-training to pre-training step-down latencies were calculated for each animal and served to index learning.Animals were exposed to a stimulus  that terminated in the onset of a mild foot shock (i.e. a US). These tone-shock (CS-US) pairings came to elicit conditioned fear responses when animals were subsequently presented with the tone. To avoid any interaction of the training context (which itself acquires an association with shock) with the CS at the time of testing, training and testing were conducted in separate distinct contexts. Two distinct experimental chambers  were used, each of which was contained in a sound- and light-attenuating enclosure. These boxes were designated as "training" and "testing" contexts, and differ as follows: The training context was brightly illuminated (100 Lux), had clear Plexiglas walls, no lick tube, and parallel stainless-steel rods  forming the floor. The test context was dimly illuminated (6 Lux), the walls covered with an opaque pattern of alternating black and white vertical stripes (3 cm wide), and the floor was formed from stainless 1.5 mm rods arranged at right-angles to form a grid of 8 mm squares. A water-filled lick tube protruded through a small hole in one wall of the test chamber, such that the tube's tip was flush with the interior surface of the wall at a point 3 cm above the floor. Upon contacting the tube, the animal completed a circuit such that the number of licks/sec could be recorded. This circuit was designed so that if an animal made continuous contact with the tube (i.e. "mouthed" the tip), the circuit recorded eight licks/sec, a rate that approximates continuous licking. In the training chamber, a 0.6 mA constant-current scrambled footshock (US) could be delivered through the grid floor. In both the training and test chambers, a 40 dB above background white noise (the CS) could be presented through speakers mounted at the center of the chambers ceiling.Water-deprived animals were acclimated to the training and test chambers by placing them each in both contexts for 20 min on the day prior to training. Training occurred in the training context in a single 30 min session during which each animal was administered a noise-shock pairing 10 and 20 min after entering the chamber. Each 10 sec noise terminated with the onset of a 500 msec footshock. At the end of the training session, animals were returned to their home cages for 60 min, after which they were re-acclimated to the test context for 20 min where they were allowed free access to the lick tubes. On the subsequent day (23-25 hours post training), animals were tested. Each animal was placed in the test context whereupon after making 25 licks, the noise CS was presented continuously until the animal completed an additional 25 licks. The latency to complete the last 25 licks during the pre-tone interval and in the presence of the tone was recorded, with an 800 sec limit imposed on the second 25 licks . With these measures, the latency to complete 25 licks in the presence of the tone CS served as our index of learned fear, and the latency to complete 25 licks prior to CS onset served as an index of basal lick rates.For this task, animals were introduced into a round pool of opaque water from which they could escape onto a hidden  platform. The latency for animals to find the platform decreases across successive trials. We have developed a protocol in which mice exhibit significant reductions in their latency to locate the escape platform within six training trials ,37. FirsA round black pool  was filled to within 24 cm of the top with water made opaque by the addition of nontoxic, water soluble, black paint. A hidden 11 cm diameter perforated black platform was in a fixed location 1.5 cm below the surface of the water midway between the center and perimeter of the pool. The pool was enclosed in a ceiling-high black curtain on which five different shapes (landmark cues) were variously positioned at heights (relative to water surface) ranging from 24-150 cm. Four of these shapes were constructed of strings of white LEDs  and included an "X" (66 cm arms crossing at angles 40° from the pool surface), a vertical "spiral" , a vertical line (31 cm) and a horizontal line (31 cm). The fifth cue was constructed of two adjacent 7 W light bulbs (each 4 cm diameter). A video camera was mounted 180 cm above the center of the water surface. These cues provided the only illumination of the maze, totaling 16 Lux at the water surface.On the day prior to training, each animal was confined to the escape platform for 300 sec. Training was conducted on the two subsequent days. On Day 1 of training, animals were started from a unique location on each of five trials. An animal was judged to have escaped from the water  at the moment at which four paws were situated on the platform, provided that the animal remained on the platform for at least 5 sec. Each animal was left on the platform for a total of 30 sec, after which the trial was terminated. Trials were spaced at 10 min intervals, during which time the animals were held in a warmed (27.5°C) opaque (5 Lux) box lined with wood shavings. On each trial, a 90 sec limit on swimming was imposed, at which time any animal that had not located the escape platform was placed by the experimenter onto the platform, where it remained for 30 sec. Animals were observed from a remote (outside of the pool's enclosure) video monitor, and the animals' performance was recorded on videotape for subsequent analysis. Day 2 of training proceeded as Day 1. However, after the last (fifth) training trial, a 2-hour retention period was begun, after which animals were tested with a "probe" trial. On the probe test, the escape platform was removed from the pool, and all animals were started from the sixth position for that day. A 60 sec test was conducted in which the animals' time searching in the target quadrant (that in which the escape platform was previously located) and non-target quadrants were recorded.An elevated maze in the form of a "+" was constructed of black Plexiglas, each of the four arms measuring with 8 × 40 cm (W × L). A 4 mm diameter food cup was located in the center of the arm 2 cm from its end. Food (a 20 mg chocolate-flavored Noyes Rodent Pellet) was located in every cup, but was accessible to the animal on in the arm designated as "west". Twenty-four cm from the end of each arm and equidistant between successive arms were 18 × 18 cm visual cues, a black (240 pt) "X", "O", and "+".Animals were adapted to the maze on Day 1. They were placed in the north start box where they were held for 30 sec, released, and allowed to explore the unbaited maze for 3 min. This process was then repeated for the east and south arms. On Day 2, animals were trained, and a food reinforcer was present in the west arm on each trial. On the first two trials , the animal was placed in the north start box for 30 sec, and was then released and allowed to explore the maze until consuming the food in the West arm. On subsequent trials, the animal was started in the east and then south arm, followed by the reverse order of arms until a total of 12 trials  were completed. On Trials 1-12, an entry into an incorrect arm terminated the trial . If mice chose the baited arm they were allowed to consume the chocolate-flavored Noyes Rodent Pellet. An ITI of 60 sec in the home cage separated each trial. Animals' choices were recorded on each trialAn elevated maze in the form of a "T" was constructed of black Plexiglas. Each of the two cross- arms measured 36 cm in length, 4 cm in width, with 10 cm walls. A 4 mm diameter food cup was located in each cross-arm, 2 cm from its end. The base of the 'T' consisted of a 14 cm start box and a 16 cm central compartment from which the cross-arms connected. The portion designated as the "start box" could be blocked with a sliding guillotine door as can the intersection between each cross-arm and the central compartment. The maze was diffusely lit from above (80 Lux).th of a fruit loop  was available at the end of each choice arm. Over four more trials, mice were forced to alternate arm entry by closing the opposite arms' guillotine door. In the forced choice arm animals obtained a reinforcer. On the subsequent day, animals were placed in the start compartment (at the base of the T), held behind the closed guillotine door for 60 sec, and then, after the door was opened, allowed to choose one arm for entry, wherein the reward was available. On subsequent trials (30 sec ITI), the animal could choose either arm, but food was available only in the arm opposite the arm reinforced on the prior trial. Incorrect choices terminated the trial. On ensuing trials, food was available in the same arm until a correct choice was made and the food was retrieved. With our adaptation and training procedures, young adult mice often begin to perform without error after 6-10 training trials. Training proceeded for 12 trials; the number of trials required by an animal prior to its initiating three consecutive correct choices was used as an index of that animal's performance.Food deprived mice learned to alternate arm choices in a "T-maze" to obtain food reinforcement. The number of training trials required before an animal learns to consistently alternate is an index of rate at which they learn the correct pattern. On the first day of training, animals were adapted to the maze, in which 1/16ps < 0.05 were considered significant.Comparisons of groups were conducted with either one- or two-factor analyses of variance (ANOVA). In experiments where a single measure of a dependent variable (e.g., "Bolli Excreted", Fig DAT, DP, SK, KL, and GH all assisted in the design of the experiments and conducted behavioral tests. JWR and LDM conceived the study and participated in the design and coordination of the study as well as drafted the manuscript. All authors read and approved the final manuscript.


Hyperplastic and neoplastic lesions of the liver occurring “spontaneously” in C3H × Y male mice were transplanted autologously and isologously. Hyperplastic lesions did not survive in the same animal or in other animals of the same strain. Carcinomas grew in the host or in isologous mice and killed the animals. Most of the lesions were hyperplastic, and few were carcinomas. It is concluded that the histologic pattern of hyperplastic and neoplastic lesions can be correlated with the results obtained on transplantation.


Blood glucose (glc) was thenmeasured weekly for 16 weeks. Data showed thatthe experimental group contained 70% euglycemicanimals (defined as glc <200mg/dL)  versus 10% ofthe control animals (P<.05) at 14 weeks. Meanweight in the treated group was greater than theuntreated group. Insulin mRNA was detected at theinjection site of all of the treated animals, but notcontrols. Complete destruction of islets was confirmedby histology ruling out the possibility ofspontaneous reversal of insulinitis. We concludethat IM delivery of the insulin gene in the NODmouse was able to prevent clinical DM up to 14weeks in a majority of treated animals. Our experimentaldata suggests that gene therapy may be analternative treatment for IDDM in the future.Using the Adeno-associated virus (AAV) as a genedelivery vehicle, we have constructed a recombinantvector containing the full length rat preproinsulingene (vLP-1). Utilizing the well described non-obesediabetic (NOD) mouse model, an experimentalgroup (n=10) of animals were intramuscularly (IM)injected with 10


During late summer and autumn 2000, a West Nile fever outbreak in southern France resulted in 76 equine clinical cases; 21 horses died. We report the results of a large serosurvey of all equines within a 10-km radius of laboratory-confirmed cases. Blood samples were obtained from 5,107 equines, distributed in groups of 1 to 91 animals. West Nile virus immunoglobulin (Ig) G antibodies were found in 8.5% of animals (n=432). Forty-two percent of the IgG-positive animals were also IgM positive. Horses living in small groups were more affected than those in large groups. The results suggest that West Nile virus is not endemic in the affected area, the Camargue; rather, sporadic outbreaks are separated by long silent periods. West Nile virus (WNV) is an arbovirus of the genus Flavivirus, family Flaviridae. Its natural transmission cycle involves birds and mosquitoes, mainly of the Culex genus. During late summer and autumn 2000, a WNV outbreak in southern France resulted in 76 clinical cases in equines; 21 horses died Culex species, C. pipiens and C. modestus are the most abundant.In France, the first reported outbreak occurred in humans and equines during the summer of 1962 in the south of the country ,6. EquinThe 2000 outbreak occurred west of Camargue , where tOn September 6, 2000, positive serologic results (immunoglobulin [Ig] G and IgM) were first found in two horse samples. Two days later, WNV infection was confirmed by detection of viral RNA in a brain biopsy Experimental studies and sequential samples collected from naturally infected horses have shown that IgM antibodies become detectable 8–10 days post-infection and persist <2–3 months . WNV neuAfter the first horse case was confirmed, a serosurvey was ordered by the animal health authorities on all equines located within a 10-km radius of laboratory-confirmed clinical cases Blood samples were taken from all the equines within 10 km of the laboratory-confirmed cases . The useFor each animal, a form was completed by a veterinarian. Along with other data , this form noted the species and breed of the animal, its age and sex, location, and the size of the group in which the animal was included on the day the sample was taken .The individual serologic results and the data on the forms were collected in a Microsoft Access database . The horses were grouped by sex  and age . Breeds were grouped into four classes according to their typical management conditions: the pure breeds , the Camargue breed , pony breeds (often living in riding stables and used every day for short rides) and other breeds . Four frequency categories  were defined for group sizes: 1–2 animals, 3–6 animals, 7–25 animals, and >25 animals.Maps were produced with MapInfo software . The geographic data describing animal locations was the name of the “commune,” the smallest administrative French subdivision, which corresponds to an English parish.Statistical analyses were done with SAS software . A bivariate analysis was first performed, crossing each variable . The chi square test was calculated for the four variables. Prevalence ratios were computed for breed and group size, using the category with the lowest prevalence as a reference. For animals groups, chi square tests and prevalence ratios were computed both at the animal level  and the group level . Finally, a logistic regression was conducted. Animals for which the age, sex, breed, or group size had not been indicated were excluded from the data set. The dependent variable of the model was the serologic status of animals, and the independent variables were their age class, sex, breed, and group size.  For each variable, the reference class was the category with the lowest prevalence.2 and covered  99 communes.Fifty veterinarians took blood samples from 5,107 equines  distributed in 1,429 groups. The 14-week serosurvey began on September 16 (week 38) and ended December 15. The survey area was approximately 2,500 kmThe age of 4,749 animals ranged from birth to 40 years. Half the animals were geldings , 32% were mares , and 20% were stallions (n=951). Of 53 breeds noted for 4,867 animals, the predominant breed was the Camargue . Group sizes ranged from 1 to 91 animals, but most of the groups  were small . The mean group size was 3.6 animals.Of the 5,107 animals, 432 were IgG positive . Almost half (n=182) were also IgM positive . The group-level seroprevalence of the positive groups was higher than the animal-level seroprevalence: 19.2% (n=274) (95% CI 17.1 to 21.2).More than 50% of the samples were taken during the first 3 weeks; 90% of the samples had been taken at the end of week 6 . IgG-pos2. However, the density in some communes is >16 animals per km2.The map of the number of serum samples per commune shows that the geographic distribution of horses is not homogeneous : fewer sThe prevalence by commune  is higheNo significant difference was found in the serologic status of animals according to their distribution by age  contained 4,597 records. The reference classes were as follows: <1 year, mares, other breeds, and >25 animals. A slight effect was found for the Camargue breed . Conversely, the main effect was attributed to the group size variable, with decreasing ORs with increasing group size: 2.18 for groups of 1–2 animals (95% CI 1.60 to 2.96) and 1.81 for groups of 3–6 animals .Belonging to a small group and, to a lesser extent, being of the Camargue breed appeared to be risk factors for seropositivity. Comparison of the eight most affected communes with the rest of the survey area showed no significant differences by age, sex, breed, and group size (data not shown). Therefore, these risk factors are not explained by an overrepresentation of Camargue horses and of small groups near the epidemic hot spot.The geographic variations of the seroprevalence show that the epidemic hot spot was not located in a wet area, but several km to the north, in a rather dry area, even though the horse density was roughly the same in both areas. Moreover, antibodies were found in 8% of the captive mallards at a large pond in the south of the perimeter, and in four magpies of 18 captured a few km to the north, near the horse epidemic hot spot Data analysis showed no age effect. Several serosurveys in human populations have shown that, when WNV circulation is endemic in a given area, the seroprevalence tends to increase with age –13. A siA breed effect was observed in that the prevalence was higher in Camargue horses. This result reflects the usual living conditions of these rugged horses, always living outside and therefore more likely to be exposed to infectious bites.Group size had two opposite effects on seroprevalence, depending on the unit used for calculation. At the group level, the increase in seroprevalence with increasing group size is a classical finding in veterinary epidemiology: assuming all animals in a given group are exposed to the same low-level probability of infection, the more animals in the group, the higher the probability that one (or more) of them become infected. At the individual level, the decrease of the seroprevalence rate with increasing group size may be the result of two factors. First, the sizes of the groups reflect different uses and management conditions of the horses: animals kept in large groups may benefit from better management practices . However, this result could also reflect a low spatial density of infectious vectors: assuming a vector does not bite all animals of a given group but only a few of them, large groups would have a protective effect. The high density of horses in the area could also help explain the absence of reported human cases as a result of a possible zooprophylactic effect of domestic animals, as pointed out by Komar et al. The survey was intended to be comprehensive for all equines located <10 km from laboratory-confirmed clinical cases. Movements of horses in and out of the area probably occurred at the beginning of the outbreak, before the restrictive measures taken by the animal health authorities were in place. Some of the Camargue horses are half-wild and live year-round in marshes; for practical reasons, these half-wild animals were not sampled. However, even if the survey was not strictly exhaustive, the 5,107-equine sample is certainly highly representative of the domestic equines in the area.Having tested only the IgG-positive sera for IgM antibodies is an obvious bias: recently infected animals (with IgM antibodies but without IgG antibodies) that did not show clinical signs would have been missed. The seroprevalence figures obtained may be underestimated for domestic equines. Belonging to the Camargue breed was identified as a seropositivity risk factor and half-wild animals that were not sampled belong to this breed; therefore, the seroprevalence rate is probably also underestimated for the whole equine population of the area .The geographic distribution of the positive results shows that, in the east part of the survey area (and to a lesser extent the west), some positive results were still found near the area boundary: more positive results may have been included if the perimeter had been larger.The serosurvey was carried out over a 14-week period; most of the samples were taken during the first 6 weeks. Because recent WNV circulation was detected (through IgM-positive results) until week 12, some animals tested at the beginning of the study were probably infected later, and the prevalence might have been higher if the study had been conducted during winter.Few serosurveys have been conducted in equines. Survey results can be usefully compared between each other in disease-endemic areas (14); however, comparison is difficult if the survey is in epidemic areas. In such areas, the seroprevalence rate depends on the definition of the survey perimeter (which varies between studies). For example, in our study, if a radius of 15 km around cases had been used to define the survey perimeter instead of 10 km, the seroprevalence rate probably would have been lower.<2 yrs) is closer to our result: this figure better reflects the yearly infection rate and thus the infection pressure.In an endemic area, Egypt in 1959 The seroprevalence rate can also be compared with the results of two earlier serosurveys conducted in the studied area. After the 1962 epidemic , in 37 sThe results of this study, the first large-scale WNV serosurvey conducted in equines, show that seroprevalence rate does not increase with age. Assuming that, as neutralizing antibodies, IgG antibodies persist several years after infection, this result (and the WNV history in the studied area since 1962) suggest that the Camargue is an epidemic area, with outbreaks that occur periodically and are separated by long silent periods. The seroprevalence level, especially for animal groups, indicates that virus circulation was intense during the 2000 epidemic. This intense virus circulation and the location of the epidemic focus in a dry area could be explained by the amplification by synanthropic bird species in dry areas, from a primary circulation in wet areas in water birds. The survey results do not allow us to assert whether this primary circulation is permanent . However, the survey results suggest that, if the primary cycle is permanent, it is restricted to small marshy areas. Two further studies, a serologic follow-up of captive ducks and a serosurvey focused on horses living in the marshy areas, will be conducted in 2001–2002; these studies should allow us to refine the epidemiologic status of this primary cycle.


The aim of this work was to assess the effect of chronic administration of protonated nanostructured aluminosilicate (NSAS) on the plasma cholesterol levels and development of atherosclerotic lesions in Apolipoprotein (ApoE) deficient mice fed a high cholesterol and high fat diet. Apolipoprotein E (ApoE) deficient mice were divided into the following treatment groups: protonated NSAS 1.4% (w/w), untreated control and 2% (w/w) stigmastanol mixed with high-cholesterol/high-fat diet. Animals were treated for 12 weeks, blood samples were withdrawn every 4 weeks for determination of plasma cholesterol and triglyceride levels. At the end of the study the aortic roots were harvested for assessment of atherosclerotic lesions. NSAS at 1.4% (w/w) and stigmastanol at 2% (w/w) treatment groups showed significant decreases in plasma cholesterol concentrations at all time points relative to the control animals. The lesion sum area in 1.4% (w/w) NSAS and 2% (w/w) stigmastanol groups were significantly less from the control animals. In conclusion, in this study, the effectiveness of chronic administration of protonated NSAS material in the reduction of plasma cholesterol levels and decrease in development of atherosclerotic lesions was demonstrated in Apo-E deficient mice model. Elevated plasma cholesterol levels have been associated with increased risk of atherosclerosis and coronary artery disease . AlthougMice, C57B1/6 B6.129P2-ApoE™1UNC, 4 week old, with homozygous deletion of the ApoE gene (apolipoprotein E knock-out) were purchased from Jackson Laboratories, USA. The Apo-E deficient mice model has been used extensively, since these mice develop severe hypercholesterolemia and atherosclerotic lesions similar to those observed in humans ,11.The protonated NSAS material was prepared as previously reported , See Add. BrieflyAll animals used in this study were cared for in accordance with the principles promulgated by the Canadian Council in Animal Care and the University of British Columbia. The research adhered to the "Principles of Laboratory Animal Care" .The Apo-E deficient mice  were divided into the following treatment groups: protonated NSAS 1.4% w/w, untreated control and 2% w/w stigmastanol. All animals received high-cholesterol/high-fat diet   for the duration of the study. The tested active compounds were incorporated into the diet. All animals were treated for 12 weeks. Blood samples were withdrawn from saphenous vein every 4 weeks and at the end of the study for determination of total plasma cholesterol and triglyceride levels. At the end of the study the animals were sacrificed and aortic arch was harvested for histopathology assessment of atherosclerotic lesions in control animals and in groups that showed statistically significant differences in their plasma lipid profile relatively to the control group. Tissue surrounding the aorta including all fat were trimmed, and frozen in liquid nitrogen. The aorta was cut in 3 consecutive slices (10 μmol/L) 5 mm above the aortic root. Slides were stained with Oil-Red-O, Movat's Pentachrome and Hematoxylin-eosin. An independent pathologist, blinded to the treatment groups scored the lesion formation based on cumulative atherosclerotic exposure area (sum area).Statistical analyses were performed using one-way ANOVA followed by Dunnett multiple comparisons test. Results were expressed as mean +/- SEM, p < 0.05 indicated a significant difference between groups.All animals based on physical appearance did not appear to have any deleterious effects from administration of NSAS at 1.4% w/w or stigmastanol at 2% w/w. The activity and behaviour of the animals were similar between all groups and consistent with the ApoE deficient phenotype. The baseline of the body weight was 19 g (average) and after 12 weeks was 33.2 (average). As a result of similar food and water consumption (data not shown) during all period of study, the body weight increased by the same amount over the experimental period in all groups (treatment and control)  compounds to reduce dietary cholesterol intestinal absorption in a rat model.Click here for file


Metacarpal lengthening in the hand is a new application for distraction neo-histiogenesis. Metacarpal lengthening with distraction helps in improvement in pinch function. Thumb lengthening is technically easy in comparison to other metacarpals. We present the operative treatment and post-operative outcome in nine patients with amputations and congenital anomalies.Nine patients underwent distraction osteogenesis for the treatment of amputations of the hand and other congenital anomalies. The dominant right hand was operated in eight cases and the left hand in one case. There were six males and three females. Improvement of function was always the aim of surgery. Age range was between 18 and 23 years. Thumb lengthening was performed in five patients and that of the index finger in four patients. Distraction started on the fifth post-operative day at the rate of 0.25 mm/day. Sensory function and bone consolidation was assessed before fixator removal.The mean duration of distraction was 51 days  and the distractor was removed at a mean of 150 days  and the bones were lengthened by a mean of 24 mm  There was improvement of function in all cases.The metacarpal lengthening by distraction histiogenesis in congenital and traumatic amputations is safe and simple method to improve pinch function of hand. Pioneered in Russia in the 1950s, the Ilizarov technique is used for the correction of various types of deformities and also for restoration of limb length. The use of distraction histiogenesis in metacarpals and metatarsals is a new concept.There are so many other highly skilled techniques like microvascular toe or tissue transplantation and pollicization that are possible,We have performed metacarpal lengthening by distraction histiogenesis with small external fixator in nine hands. We describe the operative procedures and our results.From 2001 to 2006, we treated nine hands in nine patients by distraction neo-histiogenesis. In five patients, the cause was traumatic amputation and in four patients it was congenital anomalies. Of the four, two had absence of fingers from the level of base of proximal phalanx and two were associated with syndactylism. There was no neurovascular deficit in any of the cases and the available muscle functions were normal. Length intended for lengthening was around 2 cm in all cases. Objective of lengthening was functional improvement. There were six males and three females. The right hand was involved in all cases but one. Only thumb metacarpal lengthening was performed in five patients and index finger lengthening was performed in four patients. Traumatic cases, thumb metacarpal lengthening was performed in three and index metacarpal in two cases. Age range was between 18 and 23 years. Clinical details of the patients are given in The planned rate of lengthening was 0.25 mm/day. The post-operative rate of bone lengthening was analyzed on the basis of anteroposterior radiographs made at the time of distractor removal. Regenerate, pinch function, and sensory function in the treated hand were evaluated.With the patient under brachial anesthesia, a straight incision was made on the dorsum of the first or second ray of hand and the metacarpal was exposed. The periosteum was dissected longitudinally and osteotomy was made horizontally toward the metaphysial area of the bone. A 1 mm K-wire was placed in the metacarpal to maintain alignment. The periosteum and the skin were sutured in layers. Two 2 mm K-wires were inserted 2 mm proximal and distal to the osteotomy site. The distractor device was then assembled to pins [Figures The mean duration of the distraction is 51 days (42–60 days) and of the consolidation is 90 days (70–110 days). The distraction device was removed at a mean of 150 days (140–160 days). The bones were lengthened by a mean of 24 mm (20–28 mm). On an average, a 20% increase in length was achieved. In one case there was pin loosening and in two cases non-union led to angular deformity. Hyperextension deformity of the metacarpophalangeal joint is a common complication that occurred in four of nine cases. Six months after operation, all hand movements were normal except fully active flexion of the lengthened finger. There was intact sensation of the lengthened rays. There was non-union in two cases, which were then grafted and union was achieved.The principle of distraction osteogenesis can be very effectively and rewardingly applied to the small bones of the hand. It can provide a source for enhanced prehension and gain functional length.Metacarpals were selected for lengthening due to their good vascular supply and adequate soft tissue coverage. Presence of cancellous bone and stability of the base of the metacarpal determines the level of osteotomy between base and shaft. Previous methods of lengthening by osteotomy, iliac crest grafting, and immobilization by POP cast leads to stiffness of the metacarpophalangeal joint. Distraction lengthening allows activity of the hand during lengthening as well as physiotherapy to the metacarpophalangeal joint to prevent stiffness. Bone grafting is not needed in these cases.There are few surgical options for the reconstruction of pinch function in patients with amputations of the thumb. The thumb is so small that opposition is often severely limited. Metacarpal lengthening in the thumb was adequate to allow for pinch function. The thumb metacarpal is an independent bone in comparison with other metacarpals. Hence, in our experience, thumb lengthening is easy in comparison with others.The rate of lengthening was 0.25 mm/day. On the fifth post-operative day, change of dressing was done and family members were taught pin site care as well as method of distraction. Pin site care includes once daily cleaning with hydrogen peroxide. Swabs and betadine-soaked dressings were applied. 0.25 mm distraction daily is painless and well tolerated. Patients were encouraged to use their hand during the treatment period.Consolidation was evaluated by taking antero posterior and lateral X-rays. We usually check for the formation of a minimum of three complete cortices before removal of the distractor.Pin loosening, nonunion, and metacarpophalangeal joint stiffness are some of the complications encountered. Mobilization of the metacarpophalangeal joint and active use of the hand were encouraged.Improvement of pinch and grasp functions were observed. Patients were able to write and button and unbutton dresses. Patients were operated for functional improvement. Cosmesis was never a criterion.Functional improvement by toe transfer is excellent. But, secondary operations like tenolysis, repair of ruptured tendon, tendon grafting or transfers, opponensplasty, and ligamentoplasty for joint stability were necessary in all caseset al.,Technique-wise, use of K-wire advocated by Takeshi Miyawalki et al.In children, the rate of distraction was 1 mm/dayInadequate bone formation, angular deformity, metacarpophalangeal joint stiffness, pin tract infection, and loosening are associated with this technique and care should be taken to avoid these complications.4It has been seen that inadequate bone formation is more common in the second metacarpal than the in thumb. The quality of regeneration was better in the thumb than in the second metacarpal, as seen radiologically, with no previous reports. This is probably dependant on the surrounding soft tissues. The thumb metacarpal is surrounded by muscles all around with a rich blood supply than the index finger. Careful handling of the periosteum and osteotomy in the metaphysical area prevents such complications. In this series, two cases  had poor bone formation leading to non-union and required secondary procedures in the form of antologous cancellous bone grafting. Pin tract loosening, which is a common complication, can be minimized by meticulous post-operative pin site care. We had one case of pintract loosening in index finger lengthening change pin to a more distal site (single pin only).et al.Pensler Metacarpal lengthening was found to be a simple, safe, and cost-effective method of reconstruction that provides a permanent improvement in pinch function. Holding small objects, writing, and buttoning and unbuttoning of shirts were some of the improvements in hand function post-operatively in comparison with the absence of the same functions pre-operatively.


Despite the advent of highly active anti-retroviral therapy (HAART), HIV-associated neurocognitive disorders continue to be a significant problem. In efforts to understand and alleviate neurocognitive deficits associated with HIV, we used an accelerated simian immunodeficiency virus (SIV) macaque model of NeuroAIDS to test whether minocycline is neuroprotective against lentiviral-induced neuronal injury.in vivo by proton magnetic resonance spectroscopy and post-mortem by immunohistochemistry for synaptophysin (SYN), microtubule-associated protein 2 (MAP2), and neuronal counts. Astrogliosis and microglial activation were quantified by measuring glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (IBA-1), respectively. SIV infection followed by CD8+ cell depletion induced a progressive decline in neuronal integrity evidenced by declining N-acetylaspartate/creatine (NAA/Cr), which was arrested with minocycline treatment. The recovery of this ratio was due to increases in NAA, indicating neuronal recovery, and decreases in Cr, likely reflecting downregulation of glial cell activation. SYN, MAP2, and neuronal counts were found to be higher in minocycline-treated animals compared to untreated animals while GFAP and IBA-1 expression were decreased compared to controls. CSF and plasma viral loads were lower in MN-treated animals.Eleven rhesus macaques were infected with SIV, depleted of CD8+ lymphocytes, and studied until eight weeks post inoculation (wpi). Seven animals received daily minocycline orally beginning at 4 wpi. Neuronal integrity was monitored In conclusion, oral minocycline alleviates neuronal damage induced by the AIDS virus. Early in the AIDS epidemic, severe neurological disorders including dementia were found to be caused by HIV The persistence of cumulative neurological disease despite antiretroviral therapy has led to a search for adjunctive therapies. Minocycline, an antibiotic with demonstrated neuroprotective properties, is being tested clinically in neuroAIDS . The benefits of using minocycline are multifaceted including effects against apoptotic cell death, inflammation, microglial activation in vivo1H magnetic resonance spectroscopy (1H MRS) to assess the effects of minocycline on the brain injury produced by lentiviral infection in an accelerated macaque model of neuroAIDS. The rapidly progressing simian immunodeficiency virus (SIV)-infected macaque model uses a monoclonal antibody to persistently deplete the animal of CD8+ lymphocytes in vivo1H MRS, particularly N-acetylaspartate/Creatine (NAA/Cr), are very similar to those found in people with HAND. We longitudinally examined a group of rhesus macaques infected with SIV and CD8+ cell depleted (n = 11), a subset of which received minocycline (n = 7). We demonstrate an arrest of declining NAA/Cr, indicative of restoration of neuronal metabolism, and decreases in astrogliosis, microglial activation, and neuronal loss with minocycline treatment in SIV-infected macaques.In this study, we conducted serial neuroimaging using Animals treated with anti-CD8 depleting antibody were either persistently depleted before reemergence of peripheral CD8 cells  or short-term depleted . Four MN-treated animals were persistently depleted and three were short-term depleted. Four untreated infected animals were persistently depleted. All analyses were performed on these 3 groups of animals: 1) infected, untreated, persistently CD8-depleted animals, 2) infected, MN-treated, persistently CD8-depleted animals, and 3) infected, MN-treated, short-term CD8-depleted, infected animals.7 eq/mL by 12 days post inoculation (dpi)  while no further increase in plasma viral loads was observed in the MN-treated animals after 8 dpi. The animals whose CD8 T lymphocytes rebounded before 21 dpi and that received MN treatment had 1.5–2 orders of magnitude lower viral loads throughout the course of infection. The three cohorts had different plasma viral loads at 55 dpi (p = 0.003). Both minocycline cohorts had lower viral loads compared to untreated animals at 55 dpi .After SIV inoculation, the amount of virus in the peripheral blood plasma increased rapidly in all 3 cohorts, attaining levels greater than 10p<0.001, . The plaIn vivo1H MR spectra were acquired from the parietal cortex (PC), frontal cortex (FC), basal ganglia (BG) and white matter semiovale (WM). 1H MR spectra acquired with short echo time (TE = 30 ms) are characterized by resonances primarily arising from N-acetylaspartate and N-acetylaspartylglutamate (collectively referred to as NAA), choline-containing compounds (referred to as Cho), myo-Inositol (MI), creatine-containing compounds (referred to as Cr) and the glutamate and glutamine concentrations . Consistent with previous studies myo-Inositol elevation is considered to reflect increased glial cell activity The mean percent changes in the levels of NAA/Cr as a measure of neuronal metabolism with respect to time after SIV infection in each brain region are graphically displayed in For the four SIV-infected, persistently CD8-depleted MN-treated animals (open circles), four weeks of MN treatment starting at 4 wpi resulted in a stabilization of NAA/Cr levels in all regions with partial recovery to values that were not significantly different from baseline. Treatment with MN resulted in progressive increase in NAA/Cr levels after treatment initiation in all brain regions in the three SIV-infected, short-term CD8-depleted, MN treated animals (solid triangles) .The SIV-infected untreated animals exhibited the lowest levels of NAA/Cr at the end of the study (8 wpi) in all brain regions examined . NAA/Cr The differences detected in the NAA/Cr ratio in various brain regions may be due to changes in either or both metabolites. In an attempt to identify whether changes in NAA and/or Cr are responsible for the changes observed in the ratio, NAA and Cr concentrations were estimated using tissue water as the internal standard. Due to the fact that there are over four orders of magnitude differences between the concentrations of the metabolites and water concentrations, the values have a much higher variance compared to the metabolite-metabolite ratios. Naturally, this makes obtaining statistical significance more difficult than for comparisons involving metabolite-metabolite ratios.The mean NAA concentrations in untreated SIV-infected animals declined relative to baseline measurements in each brain region analyzed, but the differences were statistically significant only in the WM . The mean Cr concentrations in these untreated SIV-infected animals were elevated in all four regions, but statistical significance was found only in the parietal cortex and white matter .The initiation of minocycline treatment four weeks after inoculation in seven animals arrested the decline in NAA and increase in Cr concentrations in the brains of these animals. The effect of minocycline treatment and the combined effects of minocycline with partial immune reconstitution of the CD8 T cell population are best appreciated when the brain concentrations of NAA and Cr at the last data point are compared in the three cohorts. As shown in Significant changes in choline (Cho) were observed in all eleven animals during the first four weeks of SIV infection (p<0.001), with an increase at 2 wpi followed by a decrease to baseline values or below, as seen in the WM. In the four SIV-infected, persistently depleted, untreated animals Cho again increased above baseline levels at 8 wpi . The increases between 4 and 8 wpi were statistically significant in the PC (p = 0.003) and WM (p = 0.003) (data not shown).In contrast, increases in Cho after 4 wpi were not significant in MN-treated, short-term CD8-depleted animals. In MN-treated, persistently CD8-depleted animals, increases in Cho after 4 wpi were not significant in the parietal cortex; however, Cho did increase significantly in the WM (p = 0.003) and in the BG (p = 0.001). Furthermore, Cho levels in the animals at their last scan before sacrifice was significantly different among the three cohorts in the PC and WM  with theMyo-Inositol (MI) levels show an increase at 2 and 4 weeks after inoculation and then normalization to baseline values (data not shown). Within the first 4 weeks of infection MI significantly increased at 2 weeks in the FC and WM  and remained elevated at 4 weeks for all eleven animals. At later time points, even without treatment, MI decreases and becomes indistinguishable from baseline levels  decreased in the parietal cortex at 2 and 4 wpi compared to baseline values (p = 0.002). The same trends are observed in the other brain regions; however, changes did not reach statistical significance. After 4 weeks Glx levels did not change significantly. The Glx levels of the three cohorts at the animals' last scan before sacrifice were not significantly different from each other. Due to the large experimental error and variance in the measurements of glutamine and glutamate, larger cohort sizes may be needed to identify any statistically significant changes.post-mortem studies a control cohort of four age-matched uninfected, CD8 T-lymphocyte depleted animals was introduced to reveal changes from “baseline”. The uninfected CD8-depleted cohort was chosen to detect the effect of minocycline on SIV infection and not on CD8 depletion. Using in vivo MR spectroscopy and post-mortem IHC, our group has previously shown that CD8 depletion without SIV infection does not change brain metabolism or provoke neuronal or glial degradation For all following Decreases in the expression of synaptophysin (SYN), an integral protein in presynaptic terminals, and microtubule-associated protein 2 (MAP2), a marker for neuronal cell bodies and dendrites, reflect neurodegeneration severity and have been found to correlate to the degree of neurocognitive impairment in persons with HIV There was a statistically significant difference in frontal cortex SYN levels among the four cohorts . There was, however, a statistically significant difference in parietal cortex neuronal counts among the four cohorts (ANOVA p = 0.017) showing decreased numbers of neurons in untreated animals compared to both the MN-treated persistently CD8-depleted animals and short-term CD8-depleted animals.Glial fibrillary acidic protein (GFAP) is the principal intermediate filament in mature astrocytes. Astrocytes rapidly synthesize GFAP in response to a neurologic insult, and it is the most commonly used marker of astrogliosis. GFAP levels were quantified in the frontal and parietal cortex of the same four cohorts of animals used for the neuronal marker determinations described in the previous section  while there was no statistically significant difference in IBA-1 positive particles/mm2 in MN-treated animals compared to the controls. In the parietal cortex, no significant differences in IBA-1 levels among the cohorts were observed.To quantify the degree of IBA-1 upregulation induced by SIV infection in CD8-depleted animals, immunohistochemistry computer image analysis was performed on brain sections from uninfected, CD8-depleted control animals along with those from the two SIV-infected, persistently CD8-depleted cohorts, of which included one cohort that was MN-treated and one untreated cohort. We did not perform computer-aided image analyses on IBA-1 for the three MN-treated, short-term depleted animals and we recognize that this was an experimental flaw in our study. in vivo1H MR spectroscopy provides an exceptional opportunity to efficiently explore drug therapies that can control neuronal injury HIV-associated neurocognitive disorders continue to be a significant problem despite the use of highly active anti-retroviral drugs (HAART), and this has motivated a search for adjunctive therapies. The SIV-infected accelerated macaque model of neuroAIDS in combination with The SIV-infected rhesus macaque shares very similar pathology with HIV-infected human patients, including the development of AIDS, disease of the CNS, and cognitive or behavioral deficits et al. reported 10% decreases in NAA/Cr in patients with mild cognitive motor dysfunction Moreover, the neuroimaging abnormalities observed in this animal model are similar to changes in HIV-infected patients that correlate with neurocognitive impairment. Chang Declines in the neuronal marker NAA and in NAA/Cr have been found to correlate with disease severity of macaque SIV encephalitis Minocycline treatment beginning at 4 wpi was found to arrest further decrease in NAA/Cr in SIV-infected, persistently CD8 T cell depleted animals. In addition, a complete recovery of NAA/Cr was observed in MN-treated animals that had partial immune reconstitution of the CD8 T cell population. The recovery of the NAA/Cr ratio was found to be due to increases in NAA, suggesting neuronal recovery, and decreases in Cr, possibly reflecting downregulation of glial and inflammatory cell activation. These salutary effects observed by brain imaging were accompanied by clinical improvement including weight gain (data not shown).post-mortem analyses. Synaptophysin (SYN) is a membrane-bound glycoprotein of the small synaptic vesicles, consistently expressed in all pre-synaptic nerve endings of the CNS The beneficial effects of MN on neuronal health were confirmed by Minocycline has been found to be neuroprotective in models of several neurological diseases including ischemia Another possible mechanism for neuroprotection is suggested by the finding of lower viral loads in MN-treated, SIV-infected macaques. Indeed, complete recovery of NAA/Cr was observed in the animals with partial immune reconstitution of the CD8 T cell population. These animals had the lowest levels of plasma viral loads of the three SIV-infected cohorts. A similar effect by MN in a different nonhuman primate model of accelerated AIDS was reported by Zink All four untreated and four of the seven minocycline-treated animals had persistent depletion of CD8+ lymphocytes. However, three of the treated animals had partial recovery of CD8+ lymphocytes. This latter cohort was distinct from the other two with respect to viral loads, imaging, neuropathology and other measures. The better profile of this cohort demonstrates the essential role of CD8+ lymphocyte depletion in producing the neuroAIDS spectrum up to SIV encephalitis. It also demonstrates that while MN treatment alone is insufficient to fully protect the brain in this model and, presumably, the brain in HIV-infected individuals, a combination of approaches may lead to superior results. Thus, the best strategy to treat neuroAIDS may be with combination therapy of minocycline and antiretroviral therapy.Some studies have found conflicting results with respect to the neuroprotective properties of minocycline in vivo1H MR spectroscopy to noninvasively assess minocycline as a neuroprotective agent in neuroAIDS and suggests that the integration of MR spectroscopy should be considered for use in clinical trials of minocycline in patients with HAND.In conclusion, short-term administration of oral minocycline resulted in significant neuroprotection in the SIV-infected macaque accelerated model of neuroAIDS. The study also demonstrated the efficacy of All animal studies were performed in accordance with federal laws and regulations, international accreditation standards, and institutional policies, including approval by the Massachusetts General Hospital Subcommittee on Research Animal Care and the Institutional Animal Care and Use Committee of Harvard University. All animals received environmental enrichment and were monitored daily for evidence of disease and changes in attitude, appetite, or behavior suggestive of illness. Appropriate clinical support was administered under the direction of the attending veterinarian and included analgesics, antibiotics, intravenous fluids, and other supportive care. Animals were euthanized when they presented with advanced stages of AIDS; criteria for euthanasia included 15% weight loss in two weeks, unresponsive opportunistic infection, persistent anorexia, intractable diarrhea, progressive neurologic signs, significant cardiac or pulmonary signs or other serious illness.Eleven 4–5 years old  rhesus macaques (Macaca mulatta) were included in this study . All ani2 and respiratory rate were monitored continuously. A heated water blanket was used to prevent hypothermia. All animals were anesthetized with ketamine-HCl and euthanized by intravenous pentobarbital overdose.Animals were scanned two times before infection and biweekly until sacrifice. For MR imaging, each animal was tranquilized with 15–20 mg/kg intramuscular ketamine hydrochloride and intubated to ensure a patent airway during the experiment. Intravenous injection of 0.4 mg/kg atropine was administered to prevent bradycardia. Continuous infusion of approximately 0.25 mg/kg/min propofol was maintained throughout imaging via catheter in a saphenous vein. Heart rate, oxygen saturation, end-tidal COpost-mortem evaluations as control cohort.One additional cohort of four uninfected CD8-depleted macaques  were included for the 1H MRS volume of interest (VOI), sagittal and axial turbo spin echo MRI were acquired at 140×140 mm2 field of view (FOV), TE = 16 ms and 512×512 matrix size. Other imaging parameters include a 2 mm slice thickness for sagittal images, a 1.2 mm slice thickness for axial images and repetition times (TR) of 4500 ms and 7430 ms for sagittal and axial images, respectively.All experiments were performed with a 3 Tesla whole-body imager  using a circularly polarized transmit-receive extremity coil. First, a three-plane localizer was performed to position the monkey in the coil. In this manner, voxel placement was highly reproducible. To image-guide the 1H MR spectroscopy was performed in the parietal cortex (PC), frontal cortex (FC), basal ganglia (BG) and white matter semiovale (WM) using a point resolved spectroscopy sequence (PRESS) with water suppression enhanced through T1 effects (WET) Single voxel All spectra were processed offline using the LCModel software package Blood and CSF were drawn before every MR scan, blood was centrifuged, and plasma and CSF were stored at −80°C until study endpoint. Virion-associated SIV RNA in plasma was measured by using a real-time reverse transcription-PCR assay on an Applied Biosystems  Prism 7700 sequence detection system with a threshold sensitivity of 100 copy eq/mL, as previously described CD8+ T lymphocyte depletion was monitored by flow cytometry prior to infection and CD8 depletion and weekly thereafter. Flow cytometric analyses were performed with 100-µl aliquots of blood incubated with fluorochrome-conjugated antibodies including anti–CD3-APC , anti–CD4-FITC , anti–CD8-PE , and anti–CD20–PE–Texas Red . Following antibody incubation for 15 minutes at room temperature, cells were washed twice with PBS containing 2% FBS; erythrocytes were lysed using ImmunoPrep Reagent System (Beckman Coulter); and samples were washed with PBS, resuspended in 2% formaldehyde in PBS and analyzed on a FACSCalibur flow cytometer (BD). The absolute number of CD8+ T lymphocytes was determined by multiplying the percentage of CD8+ CD3+ T cells by absolute lymphocyte counts obtained using a standard veterinary 3-point WBC differential, CBC Hematology Analyzer .At day of sacrifice, all animals were anesthetized with ketamine-HCl and euthanized by intravenous pentobarbital overdose. Animals were perfused with 4 liters of chilled saline, and CNS tissues were collected in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6 µm and stained with hematoxylin and eosin (H&E). All H&E sections were evaluated by a neuropathologist.Microglial activation was assessed by quantifying calcium binding adaptor protein 1 (IBA-1). Frontal and parietal cortical brain sections of the four untreated animals euthanized at 8 wpi and the four persistently CD8-depleted animals were incubated with rabbit anti-IBA-1 (Wako Corp. Japan) to determine the amount of microgliosis. In addition, frontal cortex and parietal cortex from four uninfected, CD8-depleted animals served as the control cohort for the IBA-1 analyses. Images of tissue sections were captured without manipulation using an Olympus 3-CCD T60C color video camera mounted on an Olypus Vanox-SI microscope and analyzed using NIH Image J software.Quantitative immunohistochemistry for glial fibrillary acidic protein (GFAP), synaptophysin (SYN), microtubule-associated protein 2 (MAP2) and neuronal counts was performed on fifteen animals: 1) four uninfected CD8-depleted control animals, 2) the four SIV-infected, CD8-depleted untreated animals sacrificed at 8 wpi, 3) the four MN-treated, persistently CD8-depleted SIV-infected animals and 4) the three MN-treated, short-term CD8-depleted, SIV-infected animals.The degree of reactive astrogliosis was assessed with the monoclonal anti-glial fibrillary acidic protein . 5 µm-thick paraffin sections from the frontal cortex were immunolabeled overnight with these monoclonal antibodies followed by biotinylated horse anti-mouse immunoglobulin G, avidin-horseradish peroxidase , and reacted with diaminobenzidine tetrahydrochloride and peroxide (0.03%). The integrity of the synapses was evaluated with the monoclonal antibody against synaptophysin (1∶10) . The status of neuronal dendrites was evaluated by using monoclonal antibody against microtubule-associated protein 2 (MAP2) (Boehringer Mannheim).Levels of GFAP, synaptophysin, and MAP2 were estimated by means of computer-aided image analysis, as previously described The number of cortical neurons was quantified by using stereologic evaluation. Formalin-fixed paraffin sections (7 µm thick) from the frontal and parietal cortex were stained with cresyl violet for subsequent computer-aided image analysis as previously described in vivo MR spectroscopy data and viral loads, repeated measures analysis of variance (RM-ANOVA) in combination with Holm's t-tests was employed to isolate differences between time-points within the cohorts using JMP 7.0 . For the post-mortem measures including IBA-1, SYN, MAP2, GFAP, neuronal counts and last MRS scans before sacrifice, ANOVA was performed among the cohorts and if found to be significant, Least Square Means Student's t-tests were used to isolate which cohort was significantly different. A p-value of ≤ 0.05 was considered to be significant.For the serial


Although there has been a reduction of rabies in pets and domestic animals during recent decades in the United States, rabies remains enzootic among bats and several species of terrestrial wildlife. Spillover transmission of wildlife rabies to domestic animals therefore remains a public health threatRetrospective analysis of surveillance data of reported animal incidents  from South Carolina, 1995 to 2003, was performed to assess risk factors of potential rabies exposures among human and animal victims.Dogs and cats contributed the majority  of all reported incidents, with stray dogs and cats contributing 9.0% and 15.1 respectively. Current rabies vaccination status of dogs and cats  were below World Health Organization recommended levels. Owned cats were half as likely to be vaccinated for rabies as dogs . Animal victims were primarily exposed to wildlife (83.0%), of which 27.5% were rabid. Almost 90% of confirmed rabies exposures were due to wildlife. Skunks had the highest prevalence of rabies among species of exposure animals (63.2%). Among rabid domestic animals, stray cats were the most commonly reported (47.4%).While the majority of reported potential rabies exposures are associated with dog and cat incidents, most rabies exposures derive from rabid wildlife. Stray cats were most frequently rabid among domestic animals. Our results underscore the need for improvement of wildlife rabies control and the reduction of interactions of domestic animals, including cats, with wildlife. Procyon lotor), skunks (mostly Mephitis mephitis), and foxes (mostly Vulpes vulpes) [Rabies in pets and domestic animals has been successfully reduced in the United States during recent decades with vaccination programs. Oral rabies vaccination (ORV) and other control programs in wildlife, on the other hand, have had limited success. Rabies remains enzootic among bats and several species of terrestrial wildlife ,2, inclu vulpes) . Spillov vulpes) .There is currently no national surveillance for animal exposures, including bites, scratches, or mucous membrane contact with saliva or nervous tissue ,6. SouthThis study is based on Animal Incident/Rabies Investigation Reports collected by the South Carolina Department of Health and Environmental Control (SCDHEC) between 1994 and 2004 from Appalachia I, II, and III Public Health Districts that include the seven northern counties of Anderson, Cherokee, Greenville, Oconee, Pickens, Spartanburg, and Union. Such reports relate incidents of potential rabies exposures and include all reported animal bites, scratches, or potential mucous membrane contacts. Potential rabies exposures are reported by staff of medical treatment facilities, victims or bystanders  and are investigated by environmental health specialists with SCDHEC. Animal bites to humans are included in the South Carolina List of Reportable Conditions and is therefore mandated by law and regulation. Animal bites to animals are only reported voluntarily. As we were only concerned with terrestrial rabies, we excluded incidents involving bats (N = 245). If several people receive potential rabies exposures through the same animal a separate report results for each individual. Therefore, the number of Animal Incident/Rabies Investigation Reports may be different from the number of exposure animals.Animal Incident/Rabies Investigation Reports were classified by victim type . The "no victim" category was assigned to reported observations of atypical, erratic, or aggressive behavior of an animal, which occurred with no physical injury or mucous membrane exposure. Exposure animals were defined as animals implicated in an Animal Incident/Rabies Investigation Report. Dogs and cats were categorized according to ownership status . Vaccination status according to SCDHEC Animal Incident/Rabies Investigation Reports was verified by SCDHEC staff for each case and post-exposure prophylaxis  was recorded. Stray animals, defined as those with no identified owner or individual harboring the animal, were considered unvaccinated. Similarly, dogs and cats with unknown immunization status were assumed to be unvaccinated. If a dog or a cat was classified as "stray", but was reported as having been vaccinated against rabies, it was excluded from analyses of ownership status.All counties, with the exception of Cherokee county, utilize the dBase VIPER computerized database system. A database was created for Cherokee County data for 1998 – 2003 utilizing the original hard copy Animal Incident/Rabies Investigation Report forms. Use of computerized records varied by time period for some of the counties and resulted in 9 years of data from 3 counties, 5 years of data from two counties, and 3 years of data from two counties. Published SCDHEC laboratory confirmed animal rabies reports were utilized to confirm accuracy of county data. Animal rabies was diagnosed by direct fluorescent antibody testing of submitted brain tissues. Monoclonal antibody testing was used to determine the viral variant present in each positive sample.Overall, 26 records were excluded from the analysis, because of inconsistencies, such as the specification of an animal species for the victim if the victim was human (N = 12) or no victim was reported (N = 4) or when no victim species was given for animal victims (N = 10). For rate calculations, 2000 census data was used.To quantify bivariate associations between dichotomous exposure and outcome variables, we calculated odds ratios with asymptotic 95%-confidence intervals (CI) and associated p-values. For 22 contingency tables with small cell sizes we calculated exact confidence intervals for the odds ratios. For proportions, we calculated exact binomial confidence intervals. Proportions were compared using the Chi-square test for 2 × 2 contingency tables. We used an alpha level of 0.05. All statistical analyses were performed using SAS .Overall, 22,485 animal incidence reports were included in this study. The vast majority of incidents involved human victims and accounted for 95.4% of the reports. The average annual incidence rate of incidents involving people was 203.7 per 100,000.Dogs and cats most commonly caused incidents involving people  Table . Wild anAlthough dogs were overall more commonly implicated in incident reports than were cats, stray cats  were more frequently reported than stray dogs N = 2,017)  , while in owned cats, that proportion was less than a third 32.3%)  . For animals, the most common source of rabies exposure was raccoons, followed by skunks, foxes and cat Table . For peoOur results are consistent with some previous reports indicating that the majority of reported incidents in which an animal has potentially exposed a human to rabies are associated with pet dogs -10. WhilOverall, incidents in which an animal has potentially exposed another animal or human to rabies are likely to be under-reported . Patricka priori probability of being positive. This may be a result of case selection, as skunks are difficult to catch and unpleasant to handle. Skunks that are ill, possibly as a result of rabies, may be easier caught and thus submitted for testing. Skunks have been implicated as important hosts and vectors of raccoon rabies virus [The raccoon viral variant is the endemic terrestrial variant found in South Carolina and was the only rabies variant identified in this study. Raccoons were the most frequently tested exposure animals, which supports the finding by O'Bell and colleagues  for all es virus ,18. Furtes virus  and undeThe evidence of spill-over transmission of raccoon rabies to domestic animals in South Carolina is consistent with previous studies . As a reIn our study, rabies vaccination coverage for both dogs and cats was lower than the 70% recommended by the World Health Organization (WHO) to effectively create herd immunity and prevent sustained transmission of rabies . Lack of vaccination, on the other hand, will provide an opportunity for spillover transmission to domestic animals and their human companions. Veterinary rabies immunization protects and benefits both animal and human communities. The simplicity and proven efficacy of vaccination are the foundation of rabies prevention . The incWhile the majority of reported potential rabies exposures are associated with dog and cat incidents, most rabies exposures derive from rabid wildlife. Given a reported incident, domestic animal victims are much more likely to be exposed to rabies than people. Stray cats were disproportionately associated with human potential rabies exposures and were most frequently reported rabid among domestic exposure animals. Historically, rabies control has focused on vaccination and control of stray dogs. Our results underscore the need for future research to improve wildlife rabies control methods as well as the need for initiatives to reduce interactions of domestic animals, particularly cats, with wildlife.The authors declare that they have no competing interests.CR compiled the data, initiated the data analysis and manuscript preparation and wrote the first version of the manuscript; DG supervised the data compilation and helped write and edit the manuscript; IF supervised/conducted the analysis of the data and helped write and edit the manuscript and revised the manuscript according to reviewers' suggestions.The pre-publication history for this paper can be accessed here:


The concept that oxidative stress contributes to the development of human preeclampsia has never been tested in genetically-defined animal models. Homozygous deletion of catechol-O-methyl transferase (Comt-/-) in pregnant mice leads to human preeclampsia-like symptoms  resulting from extensive vasculo-endothelial pathology, primarily at the utero-fetal interface where maternal cardiac output is dramatically increased during pregnancy. Comt converts estradiol to 2-methoxyestradiol 2 (2ME2) which counters angiogenesis by depleting hypoxia inducible factor-1 alpha  at late pregnancy. We propose that in wild type (Comt++) pregnant mice, 2ME2 destabilizes HIF-1 alpha by inhibiting mitochondrial superoxide dismutase (MnSOD). Thus, 2ME2 acts as a pro-oxidant, disrupting redox-regulated signaling which blocks angiogenesis in wild type (WT) animals in physiological pregnancy. Further, we suggest that a lack of this inhibition under normoxic conditions in mutant animals (Comt-/-) stabilises HIF-1 alpha by inactivating prolyl hydroxlases (PHD). We predict that a lack of inhibition of MnSOD, leading to persistent accumulation of HIF-1 alpha, would trigger inflammatory infiltration and endothelial damage in mutant animals. Critical tests of this hypothesis would be to recreate preeclampsia symptoms by inducing oxidative stress in WT animals or to ameliorate by treating mutant mice with Mn-SOD-catalase mimetics or activators of PHD. Approximately 5–7% of pregnant women worldwide suffer from common hypertensive pregnancy disorders culminating in preeclampsia (PE), intrauterine growth restriction and premature child birth. PE is the major cause of maternal mortality (80%) in developing nations and in recent years, the perinatal mortality and morbidity in developed countries have increased by five-fold ,2. Moreo2O2 (nano-micromolar) are necessary for angiogenesis . Thi see Fig  and inhi animals . Such doComt-/- mice, the lack of inhibition of MnSOD under normoxic conditions (late pregnancy) would facilitate untimely accumulation of H2O2 which is essential for HIF-1α stability [2O2 would block hydroxylation of HIF-1α by inactivating PHD (PHD-Fe+3) [Comt-/- mice at late pregnancy [2 O2 (> 200 μM/L) under normoxic conditions together with stable HIF1-α is sufficient to inflict vascular pathology in Comt-/- mice. Moreover, HIF-1α is a potent mediator of myeloid cell (monocytes and macrophages) infiltration at the sites of inflammation [Comt-/- mice.In tability . H2O2 woHD-Fe+3) -29 This regnancy . Increasammation  and lipoammation . Therefoet al [1. Preeclampsia symptoms in normal pregnant mice could be created by treating the animals with 2ME2. 2ME2 which peaks at third trimester of pregnancy, is a pro-oxidant and rather than an antioxidant as proposed by Kanasaki et al . Therefo2.-, the same experiment could be repeated to ensure maximum oxidative effect. The logic behind using the combination is that rotenone at low concentration (50 nM) would direct O2.- production by diverting electron flow from complex 1 of the electron transport chain to O2, while non-toxic concentrations of 2ME2 (0.3 μM) would synergistically facilitate O2.- accumulation by inhibiting SOD [2. Since 2ME2 at low concentrations (0.3 mM) in combination with rotenone  is a potting SOD .2O2 in Comt-/- mice could be rescued by treating the animals with synthetic MnSOD/catalase mimetics. Synthetic MnSOD/catalase mimetics have been shown to exhibit both SOD and catalase activities, and some are more potent, stable and cytoprotective than the native antioxidant enzyme SOD [1. The proposed oxidative damage induced by accumulation of Hzyme SOD .2. The preeclampsia phenotype in mutant animals could be rescued by treating the animals with ascorbate or specific activators of PHD .While oxidative stress has been proposed to be central to placental pathogenesis and systemic vasculo-endothelial damage in human preeclampsia and a hypertensive mouse model , the conComt): Catechol-O-methyl transferase; (2ME2): 2-methoxyestradiol 2; (HIF-1α): Hypoxia induciblefactor-1α; : Superoxide dismutase 1–3; (MnSOD): Mitochondrial superoxide dismutase; (PHD): Prolylhydroxlases; (ROS): Reactive oxygen species; (WT): wild type.(ATP): Adenosine triphosphate; (GPCR): G protein-coupled receptor; (ISP3): Inositol triphosphate; (ER): Endoplasmic reticulum; (The authors declare that they have no competing interests.The authors discussed the clinical importance of different mouse models for preeclampsia and developed the hypothesis; SB wrote the manuscript. All authors read and approved the final manuscript.