The kinematics and kinetics of riding a racehorse: A quantitative comparison of a training simulator and real horses.

Movement of a racehorse simulator differs to that of a real horse, but the effects of these differences on jockey technique have not been evaluated. We quantified and compared the kinematics and kinetics of jockeys during gallop riding on a simulator and real horses. Inertial measurement units were attached mid-shaft to the long bones of six jockeys and the sacrum of the horse or simulator. Instrumented stirrups were used to measure force. Data were collected during galloping on a synthetic gallop or while riding a racehorse simulator. Jockey kinematics varied more on a real horse compared to the simulator. Greater than double the peak stirrup force was recorded during gallop on real horses compared to the simulator. On the simulator stirrup forces were symmetrical, whereas on a real horse peak forces were higher on the opposite side to the lead limb. Asymmetric forces and lateral movement of the horse and jockey occurs away from the side of the lead leg, likely a result of horse trunk roll. Jockeys maintained a more upright trunk position on a real horse compared to simulator, with no change in pitch. The feet move in phase with the horse and simulator exhibiting similar magnitude displacements in all directions. In contrast the pelvis was in phase with the horse and simulator in the dorso-ventral and medio-lateral axes while a phase shift of 180° was seen in the cranio-caudal direction indicating an inverted pendulum action of the jockey.

Quantification of bovine oxylipids during intramammary Streptococcus uberis infection.

Streptococcus uberis mastitis results in severe mammary tissue damage in dairy cows due to uncontrolled inflammation. Oxylipids are potent lipid mediators that orchestrate pathogen-induced inflammatory responses, however, changes in oxylipid biosynthesis during S. uberis mastitis are unknown. Thus, the current objective was to determine how oxylipid concentrations change in milk and mammary tissues during different stages of S. uberis mastitis. Increased arachidonic acid and linoleic acid-derived oxylipids were significantly increased in S. uberis-infected bovine mammary tissue. Linoleic acid metabolites, hydroxyoctadecadienoic acid (HODE) and oxooctadecadienoic acid, predominated in tissue and milk. Furthermore, in vitro exposure of bovine mammary endothelial cells to 13-hydroperoxyoctadecadienoic acid, upstream metabolite of HODE, significantly increased cyclooxygenase-2 expression, but 13-HODE exposure had no effect. The findings in the current study indicate lipidomic profiling may explain some of the dynamics of inflammation during bacterial challenge, however continued research is necessary to determine sample compartments which best reflect disease pathogenesis.

Persistent regional increases in brain-derived neurotrophic factor in the flurothyl model of epileptogenesis are dependent upon the kindling status of the animal.

Brain-derived neurotrophic factor (BDNF) appears to be both regulated by and a regulator of epileptogenesis. In the flurothyl (HFE) model of kindling mice exposed to successive flurothyl trials over 8 days express a rapid, long-lasting reduction in generalized seizure threshold and a more slowly evolving change in seizure phenotype in response to subsequent flurothyl exposure. The BDNF genotype of particular mouse strains appears to influence the epileptogenic progression in this model. Thus, we hypothesized that BDNF signaling pathways are altered by flurothyl-induced seizures. Following HFE kindling, fully kindled (eight seizures) adult male C57BI/6J mice had significantly elevated whole brain BDNF levels through at least 28 days after their final seizure. Mice that received only four HFE seizures (not kindled) had elevated BDNF levels, but only at 1 day post-seizure (DPSz), while BDNF levels were not significantly altered in mice receiving just one HFE seizure at any time point studied. Regional expression patterns of BDNF in the hippocampus, hypothalamus, and frontal cortex were also elevated by one DPSz and returned to control values by 14 DPSz in mice that received four HFE seizures. No changes were seen in the cerebellum, striatum, or piriform cortex. In contrast, fully kindled mice had significantly elevated BDNF levels within the hippocampus, hypothalamus, neocortex, and striatum that remained elevated through at least 14 DPSz, while levels were unchanged in the cerebellum and piriform cortex. Regional results were confirmed using anti-BDNF immunohistochemistry (IHC). Despite changes in BDNF levels following HFE kindling, we were unable to demonstrate alterations either in full-length tyrosine kinase receptor B (TrkB) expression (Western blot and IHC) or in truncated TrkB (IHC) expression levels. Together, these data suggest a model of a positive feedback loop involving seizure activity and seizure number and persistently modified BDNF signaling pathways that influences seizure phenotypes within the HFE kindling paradigm. Thus, long-term elevations in BDNF may be responsible in part for epileptogenic processes and the development of human refractory epilepsies.

Increased mitotic activity in the dentate gyrus of the hippocampus of adult C57BL/6J mice exposed to the flurothyl kindling model of epileptogenesis.

Repeated flurothyl-induced generalized forebrain seizures result in a progressive and permanent lowering of the generalized seizure threshold in mice and an increase in the percentage of animals expressing forebrain-brainstem seizures, when rechallenged with flurothyl, after a stimulation-free period. Since this seizure paradigm serves as an excellent model for examining changes in seizure threshold and seizure propagation, we were interested in examining mitotic activity in hippocampal progenitors following flurothyl-induced epileptogenesis. In the present studies, we investigated (1). the effect of one or eight flurothyl-induced seizures on mitotic activity in the hippocampal dentate gyrus of adult mice measured by 5-bromo-2'-deoxyuridine incorporation, (2). the time course of change in hippocampal mitotic activity, (3). the cellular phenotype of these mitotically active cells, and (4). the relationship of changes in mitotic activity to changes in seizure threshold and phenotype. Significant increases in hippocampal mitotic activity were observed in mice exposed to either one or eight flurothyl-induced seizures. Increases were observed at 1 and 3 days following one seizure, and at 0, 1, 3, and 7 days following eight seizures. Confocal analyses, using neuronal and glial markers, suggest that the majority of these mitotic cells are neurons. In addition, no correlation was observed between hippocampal mitotic activity and the final seizure type that animals expressed following incubation and flurothyl retest. A significant correlation was observed between hippocampal mitotic activity and seizure threshold in flurothyl-kindled mice. Overall, these results indicate that both one and eight flurothyl-induced seizures are potent inducers of hippocampal neurogenesis in adult mice. Results further suggest that the increases in hippocampal neurogenesis are not directly related to the processes that underlie the shift in behavioral seizure phenotype, but may be involved in either the establishment or the maintenance of seizure threshold in this flurothyl model of epileptogenesis.

Improvement of visual acuity over time in patients with bilateral geographic atrophy from age-related macular degeneration.

PURPOSE: To study the improvement in visual acuity over time in patients with central scotomas. METHODS: In a prospective natural history study of geographic atrophy (GA) from age-related macular degeneration (ARMD) with annual follow-up, 36 patients with bilateral GA completed 3 years of follow-up. Protocol visual acuity (VA) measurements were performed. Scanning laser ophthalmoscopy (SLO) was performed, and the areas of GA were measured from fundus photographs. RESULTS: Six eyes of six patients with VA ranging from 20/80 to 20/500 had a VA improvement of two or more lines (mean, 3.2 lines). This was found only in the worse-seeing eyes of the patients and was contemporaneous with the deterioration in VA of the better-seeing eyes. Four of six eyes that improved in acuity had an improvement in the ability to find and hold the fixation target in an area of seeing retina, as assessed by SLO at follow-up, and a fifth eye changed from one fixation site that had little functional retina to another site. CONCLUSIONS: Spontaneous improvement in VA in eyes with bilateral GA and central scotomas may occur. It appears to be related to deterioration in VA of the better-seeing fellow eye and is associated with improvement of fixation in the worse-seeing eye. The worse-seeing eye of a patient with bilateral ARMD may have the potential for better vision than measured VA indicates. This finding may have implications for the choice of patients in treatment trials, for interpretation of long-term results, and for planning and assessment of low vision intervention.

Enlargement of atrophy and visual acuity loss in the geographic atrophy form of age-related macular degeneration.

OBJECTIVE: To describe the progression of geographic atrophy (GA) from age-related macular degeneration (AMD) with respect to visual acuity (VA) loss and enlargement of atrophy. DESIGN: A prospectively observed case series. SETTING: Tertiary retinal referral center. PARTICIPANTS: One hundred twenty-three patients with GA due to AMD who completed at least 1 year of follow-up (median follow-up, 3 years) were examined annually. METHODS: At each examination, a protocol best-corrected VA of each eye was measured, a clinical examination was performed, and color fundus photographs were taken. The areas of atrophy were drawn and measured. MAIN OUTCOME MEASURES: Visual acuity loss and enlargement of total and central atrophy. RESULTS: At baseline, median VA was poorer with larger areas of atrophy, but there was wide variation related to sparing of the fovea. Thirty-one percent of all study eyes suffered a three-line VA loss from baseline by 2 years, and 53% had a three-line loss by 4 years. Those eyes with VA better than 20/50 had the highest rate of acuity loss; 27% of these eyes had acuities of 20/200 or worse at 4 years. Visual acuity loss in the GA study eye was similar in patients with bilateral GA and in those with choroidal neovascularization in the fellow eye. Total atrophy enlarged a median of 1.8 Macular Photocoagulation Study disc areas (DA) at 2 years; atrophy within a 4-DA circle centered on the fovea enlarged a median of 0.9 DA. Two (22%) of nine patients with GA in one eye and only drusen without advanced AMD in the fellow eye developed GA in the fellow eye at 2 years. CONCLUSIONS: Geographic atrophy is associated with a significant decline in VA over time in many eyes. Areas of atrophy continue to enlarge over time, even when already large at baseline. The combination of reduced VA with enlargement of atrophy, occurring bilaterally in most patients, can lead to significant impairment of visual function.

Measuring geographic atrophy in advanced age-related macular degeneration.

PURPOSE: To present a method developed for measuring areas of geographic atrophy (GA) in advanced age-related macular degeneration, METHODS: A microfilm reader projected the 30 degrees fundus photograph of the macula. Retinal landmarks, atrophic areas, and spared areas within the atrophy were traced, without access to drawings of other years. The total atrophic area was calculated, as was the atrophy within a four-disc-area circle entered on the estimated foveal center. The configuration of the atrophy was documented. RESULTS: Avoidable sources of discrepancy included variability in peripapillary atrophy seen on the photograph, and variability seen in the extent of the field. Reproducibility studies found a median absolute difference of 0.19 Macular Photocoagulation Study disc areas (DA) in total atrophy between repeat drawings, with 75% of repeat drawings having a difference of less than 0.33 DA. For central atrophy measures, there was a median difference of 0.08 DA, with 75% of pairs having a difference of less than 0.18 DA. Features making the definition of borders of GA difficult include the presence of drusen and pigmentary alteration, a fundus in which choroidal vessels are easily visible, and variation in the appearance of GA within a single area of atrophy. CONCLUSIONS: This method provides a reliable means of measuring the size of atrophic areas in GA and will be useful for measuring longitudinal change. It may be difficult to determine whether central spared areas are present, and correlation with visual acuity and macular perimetry may be helpful.

The development of choroidal neovascularization in eyes with the geographic atrophy form of age-related macular degeneration.

OBJECTIVE: To determine the rate of developing choroidal neovascularization (CNV) in eyes with geographic atrophy (GA) from age-related macular degeneration (AMD) and the characteristics of the CNV in these eyes. DESIGN: Prospective natural history study with cohort analysis. PARTICIPANTS: One hundred fifty-two patients with GA and no CNV by fluorescein angiography in at least 1 eye, with annual follow-up. MAIN OUTCOME MEASURES: The development of CNV. RESULTS: Thirteen eyes with GA developed CNV. For patients with bilateral GA and no CNV at baseline, 2% developed CNV by 2 years and 11% by 4 years. For patients with CNV in the fellow eye, 18% developed CNV in the study eye with GA by 2 years and 34% by 4 years. The eyes that developed CNV experienced more acuity loss than did the eyes with only GA. Within the fellow eye CNV group, those study eyes with GA that had less central atrophy (and better acuity) at baseline were more likely to develop CNV. The CNV developed at a peripheral border of GA in nine eyes, in the spared foveal region in two eyes, and in both center and border in one eye. No eye developed CNV in the area of atrophy itself. The appearance of CNV was evanescent in some cases and had a final appearance of an enlarged area of GA. Twelve other eyes had hemorrhages without definite evidence of CNV; three were thought to be suspicious for CNV and the remainder were thought to be hemorrhages that may be seen in elderly patients. CONCLUSION: An eye with GA whose fellow eye has CNV is at significant risk for the development of CNV in the GA eye. A patient with bilateral GA and no evidence of CNV is at relatively low risk for developing CNV. The CNV may be evanescent and may not be detected. Intraretinal hemorrhages unrelated to CNV are relatively common in this older population.

Bidirectional transfer between electrical and flurothyl kindling in mice: evidence for common processes in epileptogenesis.

PURPOSE: This study sought to determine whether there was a transfer of seizure susceptibility between two models of epileptogenesis, electrical kindling and a newly described model of flurothyl kindling. In this study, we determined the effects of preexposure to one kindling agent on the seizure responsiveness to the other. METHODS: Mice were divided into three groups: (a) six mice (FLK) were kindled with flurothyl, rechallenged with flurothyl after a 28-day incubation phase, implanted with olfactory bulb (OB) electrodes, and electrically kindled; (b) six mice (ELK) were implanted with OB electrodes, electrically kindled to six stage 5 seizures, and given one flurothyl trial 3 days later and a second flurothyl trial after a 28-day incubation period; and (c) six mice (IMP) were implanted with OB electrodes, tested with flurothyl at the same times as the ELK group, and later electrically kindled. RESULTS: Mice that were previously kindled with flurothyl (FLK) had significantly faster electrical kindling rates to one stage 5 seizure or to six stage 5 seizures compared with animals in the ELK and IMP groups. Mice that were previously exposed to either electrical kindling or flurothyl kindling had significantly diminished latencies to generalized seizure onset (flurothyl-induced seizure thresholds) either before or after a 28-day incubation period compared with the IMP control mice. In addition, both the FLK and ELK groups had significantly increased percentages of mice expressing forebrain-brainstem seizures, compared with the IMP group, following either rechallenge with flurothyl after a 28-day incubation or focal electrical kindling. CONCLUSIONS: These findings indicate a near-complete bidirectional transfer between these electrical and flurothyl kindling models. Mice that were previously exposed to either electrical or flurothyl kindling have increased seizure susceptibilities and altered seizure phenotypes when exposed to the other seizure paradigm. Overall, these studies indicate that previous seizures are the critical determinant of the bidirectional transfer of seizure susceptibility observed, and not the electrical or pharmacologic properties of the original kindling agent. Finally, the observation of near identity in transfer characteristics between electrical and flurothyl kindling models suggests that the proepileptogenic processes initiated by exposure to either model are similar.

The role of the ventromedial nucleus of the hypothalamus in epileptogenesis.

Flurothyl kindling initiates a time-dependent process that results in a facilitated propagation from the forebrain to the brainstem seizure system and in an increase in the complexity of behavioral seizure expression. We investigated the involvement of the ventromedial nucleus of the hypothalamus (VMH) in mediating this facilitated propagation between these seizure systems. Bilateral ibotenic acid lesions of the VMH, but not the dorsomedial nucleus of the hypothalamus (DMH), resulted in a disruption in the propagation of seizure activity from the forebrain to the brainstem. Moreover, VMH lesioned mice were able to express brainstem seizures following minimal corneal electroconvulsive shock (mECS). Together, our results indicate that the VMH is a critical substrate involved in propagating seizure activity between the forebrain and brainstem, but is not involved in the expression systems necessary for forebrain or brainstem seizure manifestations.

A role for the bilateral involvement of perirhinal cortex in generalized kindled seizure expression.

The perirhinal cortex (PRh) has been suggested as a substrate for the expression of generalized clonic seizures in the late stages of kindling development (stages 4-5). Using the induction of Fos as a marker of neuronal activation, the PRh region was investigated after kindling or nonkindling electrical stimulation. Nonkindling electrical stimulation of the PRh elicited stimulus-locked behaviors, without afterdischarge. These behaviors were characterized by rearing and bilateral forelimb clonus which were terminated upon electrical stimulus offset in half of the rats displaying this behavior (with the other half expressing self-sustained seizures). In these animals, Fos immunoreactivity was found throughout neocortical and subcortical structures in the hemisphere ipsilateral to the stimulating electrode. By contrast, Fos-immunoreactivity in the contralateral hemisphere was localized primarily in the PRh and frontal motor cortex. Likewise, similar patterns of Fos immunoreactivity were observed in both hemispheres of rats following kindling to one generalized clonic seizure from several limbic and paleocortical structures. These results suggest that the bilateral involvement of the PRh is critical in producing the bilateral behaviors associated with generalized clonic seizure expression. In support of this interpretation, infusion of 3 M KCl directly into the contralateral PRh of rats kindled to a single stage 4-5 (generalized clonic) seizure from the ipsilateral amygdala reduced seizure manifestations from a generalized clonic seizure (stage 4-5) to a unilateral clonic seizure (stage 3) without affecting measures of focal excitability. Taken together, these data indicate a role for the bilateral involvement of the PRh in generalized clonic seizure expression whether evoked from the naive or kindled state. These results further indicate that bilateral behaviors require the bilateral involvement of the structures necessary for the expression of these behaviors.

Regional analysis of the spatial patterns of Fos induction in brain following flurothyl kindling.

We have recently demonstrated that eight, daily flurothyl-induced generalized clonic seizures, followed by a four week stimulus-free interval, results in a long-lasting reduction in generalized seizure threshold and a change in the type of seizure expressed in response to flurothyl from clonic to tonic. There is a progressive increase in the probability that a mouse will express a tonic seizure during the four week interval, suggesting that prior flurothyl seizures initiate a proepileptogenic process that requires time to develop. In this study, the immunohistochemical detection of the c-fos protein (Fos) was used to evaluate whether seizure-induced epileptogenesis resulted in regional differences in the degree of neuronal activation. Fos immunoreactivity was examined 1.5 h following either a single generalized seizure, the last of eight consecutive daily seizures or a retest seizure evoked two weeks after the last of eight seizures. In each condition, generalized seizure behaviours were elicited in C57BL/6 mice using flurothyl and classified as either "forebrain" (face and forelimb clonus) or "brainstem" (running/bouncing, treading, tonic extension). The spatial distribution of Fos induction was compared on the basis of the seizure phenotype and the seizure history. The predominant differences in Fos distribution were found to be related to the type of seizure expressed regardless of the seizure history. Furthermore, the different motor components that make up a "brainstem" seizure could not be distinguished by the pattern of Fos labelling suggesting that multiple convulsive behaviours are mediated by one anatomical system. Finally, Fos induction in the ventromedial hypothalamic nucleus preceded and predicted the change in seizure type from "forebrain" to "brainstem". These data support the concept that separate anatomical systems mediate the expression of the two generalized seizure phenotypes. In addition, the ventromedial nucleus of the hypothalamus may be a point of interaction between the systems and may play a role in seizure-induced neural reorganization.

Decreased brainstem seizure thresholds and facilitated seizure propagation in mice exposed to repeated flurothyl-induced generalized forebrain seizures.

We recently have described a novel model of epileptogenesis utilizing the inhalant chemoconvulsant, flurothyl (Applegate et al., 1997; Samoriski and Applegate, 1997). The hallmark feature of this model is a change in behavioral seizure phenotype from a forebrain seizure, observed during the initial flurothyl exposures, to a brainstem seizure, elicited by flurothyl, after a 28-day stimulation free incubation period. In this study, we sought to establish the basis for this change in behavioral seizure response. To this end, we examined the effects of exposure to this paradigm on the generalized brainstem seizure threshold and on the propagation of forebrain seizures onto the brainstem seizure substrate. Ten mice were given flurothyl-induced generalized forebrain seizures on 8 consecutive days (induction phase). The other ten mice were not exposed to the flurothyl induction paradigm and served as controls. Minimal corneal electroconvulsive shock (mECS--20 mA) was used to assay whether there was any change in the animals' generalized brainstem seizure thresholds at 3, 14 and 28 days following the last flurothyl seizure trial. Mice that were exposed to flurothyl exhibited a progressive increase in the percentage of animals having a mECS-induced brainstem seizure when tested at 3 (40%), 14 (70%) and 28 (90%) days following the last flurothyl seizure. Control mice rarely had a brainstem seizure at any of the three time points tested, mostly forebrain seizures were observed. These results suggest that there is a significant progressive lowering of the brainstem seizure threshold, during the incubation phase of the flurothyl paradigm, which is coincident with the previously reported time course of change in the behavioral seizure phenotype observed using this flurothyl model (Applegate et al., 1997; Samoriski and Applegate, 1997). Following mECS testing, mice were implanted with bipolar electrodes and kindled from the olfactory bulb (OB). Mice exposed to the flurothyl paradigm demonstrated significantly faster kindling rates, longer afterdischarge durations. and longer durations of and latencies to stage 5 seizures compared to controls. Furthermore, animals exposed to the flurothyl protocol demonstrated an increase in the expression of brainstem seizures after focally-elicited OB afterdischarges. These results suggest that there is an increased interaction between the forebrain and brainstem seizure systems after exposure to this model of epileptogenesis. Together, results indicate that the change in behavioral seizure phenotype observed following exposure to our flurothyl paradigm are promoted by both decreases in brainstem seizure thresholds and facilitated forebrain seizure propagation onto the brainstem seizure system.

Global increases in seizure susceptibility in mice lacking 5-HT2C receptors: a behavioral analysis.

Previous studies have shown that mice bearing a targeted disruption of the 5-HT2C receptor gene exhibit an epilepsy syndrome associated with sporadic spontaneous seizures that occasionally result in death. In this study, we have defined the seizure susceptibility profiles of these 5-HT2C receptor mutant mice backcrossed onto a C57BL/6 background. Wild-type and mutant animals were either electrically kindled from the olfactory bulb, exposed to corneal electroshock, or tested with the chemoconvulsant, flurothyl. In all paradigms, mice lacking the 5-HT2C receptor were significantly more seizure susceptible than wild-type controls. Results indicate that mutants have lower focal seizure thresholds, increased focal seizure excitability, and facilitated propagation within the forebrain seizure system. Mutants also exhibit lower generalized seizure thresholds for the expression of both generalized clonic and generalized tonic seizures. Importantly, the 5-HT receptor antagonist, mesulergine (2 or 4 mg/kg), administered prior to electroshock testing, recapitulated the mutant phenotype in wild-type mice. Together, these data strongly implicate a role for serotonin and 5-HT2C receptors in the modulation of neuronal network excitability and seizure propagation globally, throughout the CNS.

Visual function abnormalities and prognosis in eyes with age-related geographic atrophy of the macula and good visual acuity.

PURPOSE: Geographic atrophy (GA) may cause significant compromise of visual function, even when there still is good visual acuity (VA), because of parafoveal scotomas and foveal function abnormalities antedating visible atrophy. This study evaluates the visual function abnormalities at baseline and the 2-year worsening of VA and reading rate for eyes with GA compared with a group of eyes with drusen only. METHODS: Seventy-four eyes with GA and VA greater than or equal to 20/50 from a prospective natural history study of GA were included, as were 13 eyes with only drusen. Baseline visual function testing and 2-year VA and maximum reading rate are reported. RESULTS: The worsening of VA in decreased luminance and foveal dark-adapted sensitivity showed severe abnormalities for the GA group. Contrast sensitivity was significantly reduced for the eyes with GA. Half the eyes with GA, but none of the drusen eyes, had maximum reading rates below 100 words per minute. A scanning laser ophthalmoscope (SLO) measure of the scotoma near fixation combined with a measure of residual foveal function accounted for 54% of the variability in maximum reading rate in the eyes with GA. Of 40 eyes with GA observed for 2 years, half lost greater than or equal to 3 lines of VA and one quarter lost greater than or equal to 6 lines. The nine eyes with drusen with follow-up had no significant change in VA. Low foveal dark-adapted sensitivity, SLO measures of the scotoma within 1 degree of fixation, and low maximum reading rate were statistically significant risk factors for doubling of the visual angle. Significant reduction in maximum reading rates at 2 years was present for the eyes with GA. CONCLUSIONS: The eyes with GA with good VA have profound decreases in visual function, particularly in dim lighting and in reading. Half the eyes with GA had doubling in visual angle at 2 years after the baseline examination, whereas the drusen eyes remained essentially unchanged. Impaired visual function at baseline was predictive of an adverse outcome for the eyes with GA.

Repeated generalized seizures induce time-dependent changes in the behavioral seizure response independent of continued seizure induction.

This study examined both the acute and long-lasting changes in seizure susceptibility that occur in response to the repeated induction of generalized seizure activity. Daily flurothyl-induced generalized clonic seizures resulted in a progressive decrease in both the generalized seizure threshold and the latency to the first myoclonic jerk. The threshold reduction was significant as early as the second trial and was maximal by trial 5. However, a minimum of eight seizures was necessary for the maximal reduction to be long-lasting. The present study also examined the effects of the number of seizures and the duration of the stimulation-free interval on the type of generalized seizure expressed. During the induction phase of the experiment, only generalized clonic seizures ("forebrain seizures") were expressed. If, however, the animal was retested after a 1, 2, 3, or 4 week stimulation-free interval, a progressive increase in both the proportion of animals expressing "brainstem seizure" behaviors and the median seizure score was observed. The progression of flurothyl-induced generalized seizure behaviors was significantly altered if (1) a minimum of eight generalized clonic seizures had been expressed, and (2) a minimum of a 2 week stimulation-free interval followed. Fewer generalized clonic seizures failed to reliably produce changes in seizure phenotype, even after extended stimulus-free intervals. These data indicate that specific kindling processes are initiated during the interval of repeated seizure induction and evolve in the absence of continued seizure induction. Furthermore, these mechanisms of epileptogenesis were found to be manifest predominantly as a change in the seizure phenotype expressed and to proceed independent of changes in the generalized seizure threshold.

Effects of valproate, phenytoin, and MK-801 in a novel model of epileptogenesis.

PURPOSE: We have developed and characterized a novel model of epileptogenesis based on the convulsive actions of flurothyl in mice. The hallmark feature of this model is a reliable change in the type of seizure expressed in response to flurothyl from generalized clonic to generalized tonic seizures. The purpose of our study was to evaluate the effects of chronic administration of valproate (VPA), phenytoin (PHT), and MK-801 on the change in seizure phenotype observed in our model system. METHODS: Male C57BL/6J mice received flurothyl seizures on 8 consecutive days. Two hours after the last generalized seizure, chronic drug or vehicle was administered twice daily at 12-h intervals for 28 days. The drugs evaluated were VPA (250 mg/kg), PHT (30 mg/kg), and MK-801 (0.5 mg/kg). After a 7-day drug washout period, mice were retested with flurothyl. RESULTS: Among uninjected or vehicle-injected control mice, there was a significant increase in the proportion of animals expressing tonic seizures after the 28-day stimulation-free interval. Chronic administration of VPA or MK-801, but not PHT, blocked the characteristic change in seizure type from clonic to tonic. CONCLUSIONS: The change in seizure phenotype observed after exposure to our paradigm indicates a fundamental reorganization in the propagation of flurothyl-initiated seizures. As in electrical kindling, VPA and MK-801 are effective at blocking or retarding the reorganization, whereas PHT is not. The concordance in pharmacologic profiles between kindling and our model suggests that the processes underlying changes in seizure susceptibility in these two models share mechanisms in common.

Apoptotic and necrotic cell death following kindling induced seizures.

The study was designed to determine which type of cell death occurs following kindling induced seizures, and to determine which neurons die. For this purpose seizures were kindled from the entorhinal cortex. Following a range of 5-85 stage 5 seizures, rats were sacrificed, and the tissue was prepared for analysis. The TUNEL and silver impregnation methods were used to identify apoptotic or necrotic cell death, respectively. These methods were subsequently combined with immunocytochemistry, to determine if diseased neurons expressed somatostatin or the NMDA receptor (NMDAR1). The tissue analysis demonstrated that following kindling induced seizures, 1) hippocampal and extrahippocampal neurons die, 2) some neurons die through apoptosis, others through necrosis, and 3) some of the diseased neurons express somatostatin, others the NMDAR1 and that both subpopulations of neurons are present at hippocampal and extrahippocampal sites.

Differential spatial patterns of Fos induction following generalized clonic and generalized tonic seizures.

The expression of generalized clonic and generalized tonic seizures has been suggested to result from the activation of different and independent neuronal circuits. Using the induction of the c-fos protein (Fos) as a marker of neuronal activity, we identified brain structures that are differentially associated with the expression of electroconvulsive shock-induced generalized clonic and generalized tonic seizures. Expression of either seizure phenotype resulted in a similar bilaterally symmetrical increase in Fos immunoreactivity in many forebrain structures, including the bed nucleus of the stria terminalis, hippocampal dentate gyrus, amygdala, and piriform cortex, compared to controls. However, following tonic hindlimb extension (THE), the degree of labeling in specific thalamic, hypothalamic, and brain stem areas was significantly greater than that of either controls or animals exhibiting clonic seizures. While a greater number of neurons in the hypothalamus (e.g., ventromedial nucleus), subparafascicular thalamic nucleus, peripeduncular area, deep medial superior colliculus, dorsal and lateral central gray, and paralemniscal nuclei were robustly labeled following THE, noticeably fewer cells were immunoreactive following face and forelimb clonic seizure behaviors. These differences were also found to be independent of the stimulus magnitude. In animals stimulated with the same current intensity but expressing either of the two seizure phenotypes, the pattern of Fos induction was consistent with the seizure phenotype expressed. These results demonstrate that specific subsets of neurons are differentially activated following the expression of different generalized seizure behaviors and that activity in discrete mesencephalic and diencephalic structures is more frequently associated with the expression of generalized tonic seizures than with the expression of generalized clonic seizures.

Effects of generalized convulsive seizures on corticotropin-releasing factor neuronal systems.

Most stressors generate a set of endocrine and neural adaptations that form a stress response. The corticotropin-releasing factor neurons of the paraventricular nucleus of hypothalamus integrate endocrine and neural inputs, and cause a cascade of events with resultant increased levels of pituitary adrenocorticotropic hormone and adrenal hormones. Although activation of the hypothalamic-pituitary-adrenal axis is associated with a large variety of stressors, the effects of seizures on hypothalamic corticotropin-releasing factor neurons are essentially unknown. The goal of the present study was to elucidate the effects of generalized convulsive seizures on distinct and separate corticotropin-releasing factor cell populations in brain. Seizure-activated neurons were identified immunocytochemically through their expression of the Fos protein. Seizures were induced by intraperitoneal injection of kainic acid. In the paraventricular nucleus, the vast majority of corticotropin-releasing factor-like parvocellular neurons also expressed Fos-like protein following seizure elicitation. This response was specific to corticotropin-releasing factor neurons of the paraventricular nucleus, as corticotropin-releasing factor neurons in central nucleus of the amygdala or bed nucleus of the stria terminalis did not simultaneously localize Fos following seizures.