Rich W. Clough

Effect of 5,7-dihydroxytryptamine on audiogenic seizures in genetically epilepsy-prone rats

To further assess the role of 5-HT in the modulation of audiogenic seizures (AGS) in the Genetically Epilepsy-Prone Rat (GEPR), changes in AGS severity after widespread chronic depletion of brain 5-HT by intracerebroventricular administration of 5,7-dihydroxytryptamine (5,7-DHT) were examined in moderate seizure GEPRs (GEPR-3s). Following treatment with 5,7-DHT (150 micrograms/30 microliters), a significant increase in seizure severity was observed at 2, 3 and 4 weeks as compared to vehicle-injected controls. The increase in seizure severity was evidenced by a significant increase in the incidence of tonic convulsions in 5,7-DHT treated animals (53% in treated animals compared to 0% in vehicle treated controls) over the testing period. Interestingly, the latency to wild running was increased in 5,7-DHT treated GEPRs, suggesting that depletion of brain 5-HT may slow initiation of AGS. Neurochemical analysis revealed marked depletion of 5-HT in the cortex (-96%), hippocampus (-94%), thalamus (-80%), hypothalamus (-62%), midbrain (-51%) and pons-medulla (-52%) in animals that received 5,7-DHT. However, no significant reductions in brain norepinephrine content were observed in any of the regions assayed due to the pretreatment of all animals with protriptyline. The present findings lend further support for an inhibitory action of brain 5-HT on audiogenic seizures in GEPRs.

Vagus nerve stimulation may protect GABAergic neurons following traumatic brain injury in rats: An immunocytochemical study.

Seizures and subclinical seizures occur following experimental brain injury in rats and may result from inhibitory neuron loss. This study numerically compares cortical and hippocampal glutamic acid decarboxylase (GAD) positive neurons between sham fluid percussion injury (FPI), FPI with sham Vagus Nerve Simulation (VNS), and FPI with chronic intermittent VNS initiated at 24 h post FPI in rats. Rats (n=8/group) were prepared for immunocytochemistry of GAD at 15 days post FPI. Serial sections were collected and GAD immunoreactive neurons were counted in the hippocampal hilus and two levels of the cerebral cortex. Numbers of quantifiable GAD cells in the rostral cerebral cortices were different between groups, both ipsilateral and contralateral to the FPI. Post hoc analysis of cell counts rostral to the ipsilateral epicenter, revealed a significant 26% reduction in the number of GAD cells/unit area of cerebral cortex following FPI. In the FPI-VNS group, this percentage loss was attenuated to only an 8.5% reduction, a value not significantly different from the sham group. In the contralateral side of the rostral cerebral cortex, FPI induced a significant 24% reduction in GAD cells/unit area; whereas, the VNS-treated rats showed no appreciable diminution of GAD cells rostral to the contralateral epicenter. Hippocampal analysis revealed a similar reduction of GAD cells in the FPI group; however, unlike the cortex this was not statistically significant. In the FPI-VNS group, a trend towards increased numbers of hilar GAD cells was observed, even over and above that of the sham FPI group; however, this was also not statistically significant. Together, these data suggest that VNS protects cortical GAD cells from death subsequent to FPI and may increase GAD cell counts in the hippocampal hilus of the injured brain.

Testing knowledge of human gross anatomy in medical school: An applied contextual-learning theory method

The traditional gross anatomy laboratory experience, with modifications in evaluations that we outline later, meets the criteria of contextual‐learning theory, expands the repertoire of core objectives we identify for our students, and may increase the likelihood of cognitive permanence of anatomical data. Our subjects included approximately 54 first‐year medical students from each of three sequential class years (1996, 1997, 1998). As an alternative to more typical written and practical exams, examinations in a major portion of our gross anatomy program consist of two approximately 30 minute oral expositions by each student to his or her peers and a faculty member. Students demonstrate specific detail on cadaver, x‐ray, cross sections, or a model. Clinical applications, spatial relationships, nomenclature, and functions are strongly emphasized. The results of this teaching approach to the utilization of anatomical knowledge in clinical situations requires further assessment; however, new attributes have been afforded our students with implementation of the present program: First, students learn anatomical detail equally well as the students of the more traditional system (based on board exam results). Second, students who completed the program indicate that this approach provides a useful simulation of what is expected later in their training. Third, students gradually gain confidence in verbal presentation, they demonstrate cognitive synthesis of separate conceptual issues, they retain information, and they are quite visibly more enthusiastic about anatomy and its importance in medicine. Our program demonstrates that the learning of applicable human anatomy is facilitated in a contextual‐learning environment. Moreover, by learning anatomy in this way, other equally beneficial attributes are afforded the medical student, including, but not limited to, increases in communication skills, confidence in verbal presentation, synthesis of anatomical concepts, appreciation of the clinical importance of anatomy, and the general development of professionalism.

Cortical edema in moderate fluid percussion brain injury is attenuated by vagus nerve stimulation

Development of cerebral edema (intracellular and/or extracellular water accumulation) following traumatic brain injury contributes to mortality and morbidity that accompanies brain injury. Chronic intermittent vagus nerve stimulation (VNS) initiated at either 2 h or 24 h (VNS: 30 s train of 0.5 mA, 20 Hz, biphasic pulses every 30 min) following traumatic brain injury enhances recovery of motor and cognitive function in rats in the weeks following brain injury; however, the mechanisms of facilitated recovery are unknown. The present study examines the effects of VNS on development of acute cerebral edema following unilateral fluid percussion brain injury (FPI) in rats, concomitant with assessment of their behavioral recovery. Two hours following FPI, VNS was initiated. Behavioral testing, using both beam walk and locomotor placing tasks, was conducted at 1 and 2 days following FPI. Edema was measured 48 h post-FPI by the customary method of region-specific brain weights before and after complete dehydration. Results of this study replicated that VNS initiated at 2 h after FPI: 1) effectively facilitated the recovery of vestibulomotor function at 2 days after FPI assessed by beam walk performance (P<0.01); and 2) tended to improve locomotor placing performance at the same time point (P=0.18). Most interestingly, results of this study showed that development of edema within the cerebral cortex ipsilateral to FPI was significantly attenuated at 48 h in FPI rats receiving VNS compared with non-VNS FPI rats (P<0.04). Finally, a correlation analysis between beam walk performance and cerebral edema following FPI revealed a significant inverse correlation between behavior performance and cerebral edema. Together, these results suggest that VNS facilitation of motor recovery following experimental brain injury in rats is associated with VNS-mediated attenuation of cerebral edema.

Morphological deficits in noradrenergic neurons in GEPR-9s stem from abnormalities in both the locus coeruleus and its target tissues.

The epileptic condition of the genetically epilepsy-prone rat (GEPR) appears to be caused partially by deficiencies in the locus coeruleus (LC) innervation of the superior colliculus (SC). Previous studies provide quantitative documentation of noradrenergic morphological deficits in the moderately epileptic GEPR-3. The present findings extend these studies by applying cell culture methodology to assessments of the severely epileptic GEPR-9. Our data show that total neurite length, the number of neurite branch points per cell, the cross-sectional area of cell bodies, and the cell perimeter are deficient in noradrenergic neurons in LC + SC cocultures derived exclusively from GEPR-9s compared to analogous cocultures obtained solely from nonepileptic control rats. Partial restoration of LC neuron morphology toward normal occurs when the GEPR-9 SC component of the coculture is replaced with nonepileptic control SC. Finally, when the GEPR-9 SC is cocultured with the control LC, a partial morphological deficit occurs in the otherwise normal noradrenergic neurons. However, the magnitude of this deficit is less than that observed in noradrenergic neurons of the GEPR-9 LC cocultured with the control SC. These data support the hypothesis that the developmental deficiencies of noradrenergic neurons of the GEPR-9 are derived from two sources, the LC and its target tissue, in this case, the SC. Also, intrinsic abnormalities of the LC appear to make a more pronounced contribution to the noradrenergic deficits than do those which reside in the SC.

Fos in locus coeruleus neurons following audiogenic seizure in the genetically epilepsy-prone rat : comparison to electroshock and pentylenetetrazol seizure models

Seizures in genetically epilepsy-prone rats (GEPRs) may result from hypoactivity of locus coeruleus (LC) neurons during seizures. This study examined Fos-like-immunoreactivity (FLI) in the LC following audiogenic seizures in two strains of GEPRs (GEPR-9s and -3s), and following pentylenetetrazol (PTZ) or maximal electroshock seizures (MES) in normal rats. After tonic seizure, GEPR-9s showed an identical LC-FLI response to that of normal rats following tonic seizures induced by either PTZ or MES. GEPR-3s, having clonic seizures, had less FLI in the LC. Therefore, stimulus-transcription coupling in the GEPR LC is apparently normo-typic in its FLI response to seizure and thus is not likely the root cause of NE abnormalities in this seizure model.

Neurite extension of developing noradrenergic neurons is impaired in genetically epilepsy-prone rats (GEPR-3s): an in vitro study on the locus coeruleus

A primary determinant of seizure susceptibility and severity in genetically epilepsy-prone rats (GEPRs), is a generalized deficiency in the central noradrenergic system of these animals. In particular, this deficiency includes reduced numbers of norepinephrine (NE) synaptic terminals in several brain areas and distinctly fewer NE axons within the auditory tectum. Two strains of GEPRs have been developed: GEPR-3s that have moderately severe clonic seizures and GEPR-9s that have severe tonic seizures culminating in complete hindlimb extension. Seizures in animals of each substrain are preceded by a brief episode of wild running. The developmental profile of NE axonal growth in GEPRs compared to control rats is not known, but may be causally related to NE deficiencies in this seizure model. The present study compared developmental neurite extension of fetal NE neurons in vitro between GEPR-3s and Sprague-Dawley control rats, the strain from which GEPR-3s were originally derived. Neurite arborization of individual NE neurons was assessed by quantitative morphometry following immunocytochemical identification of tyrosine hydroxylase (TH). Preliminary studies using explant and dispersed-cell cultures of control-rat tissues showed that optimal culture parameters to support neuritogenesis of LC neurons included the use of dispersed-cell cultures, Pronectin-F substrate, day-14 gestation donor-tissue, no use of cytosine-arabinofuranoside (ARA-c, a glial mitotic inhibitor) and the presence of co-cultured tectal tissue. Compared to fetal control-rat NE neurons co-cultured with fetal control-rat tectum, NE neurons derived from fetal GEPR-3 LC in co-culture with GEPR-3 tectum exhibited only 30% of the neurite extension of control-rat LC neurons and GEPR-3 LC neurons had a similarly deficient amount of branching. This study suggests, but does not prove, that deficiency in tectal NE in GEPR-3s involves a developmental deficiency in neurite extension from GEPR-3 LC neurons. Hypothetically, this deficiency may also contribute to the well described NE deficiency in other regions of the adult GEPR brain.

Expression of Fos in the superior lateral subdivision of the lateral parabrachial (LPBsl) area after generalized tonic seizures in rats

Generalized tonic-clonic seizures of brain stem origin in rats are associated with acute induction of neuronal Fos in several discrete regions of the brain. One particular site in the dorsal pons shows remarkable Fos induction following generalized tonic seizures induced by maximal electroshock in normal rats or by audiogenic stimulation in genetically epilepsy-prone rats (GEPRs). Although this area shows the most intense Fos induction of any brain area following generalized tonic seizures, its identity has been uncertain. Based on its general location, we hypothesized that this nucleus was either 1) a component of the pedunculopontine tegmentum nucleus-pars compacta (PPTn-pc) or 2) the superior lateral subnucleus of lateral parabrachial area (LPBsl). The present study used Fos-protein immunocytochemistry in combination with the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, cholecystokinin (CCK) immunocytochemistry, and neuronal tract-tracing to determine the identity of this cluster of Fos-immunoreactive neurons in the dorsal pons. Following maximal electroshock seizure (MES), Fos labeling was compared to NADPH diaphorase staining (a marker for cholinergic neurons of the PPTn-pc); retrograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) injected into the ventromedial nucleus of the hypothalamus (VMH; to identify the LPBsl) or CCK immunoreactivity (also a marker for LPBsl neurons). Results showed this cluster of Fos immunoreactive (FI) neurons to be closely associated, but not overlapping, with the lateral and most caudal aspect of the PPTn-pc. Alternatively, WGA-HRP retrograde-labeled neurons corresponded precisely with the seizure-induced FI neurons. Additionally, the location of CCK immunoreactive neurons directly overlapped with the FI neurons, although they were not nearly as prevalent. These results demonstrate that the seizure-induced FI neurons in this area are neurons of the LPBsl and not cholinergic neurons of the PPTn-pc. This is the first report of seizure-induced Fos expression specifically localized to the superior lateral subnucleus of the lateral parabrachial area.

Z-Bisdehydrodoisynolic acid (Z-BDDA): an estrogenic seco-steroid that enhances behavioral recovery following moderate fluid percussion brain injury in male rats.

Several lines of research suggest that estrogens (and estrogenic compounds) are neuroprotective following experimental traumatic brain injury. However, therapeutic use of estrogens in this and other regards remains controversial. Therefore, analysis of estrogen-like compounds without potential problems similar to estrogens seems warranted. (±) Z-Bisdehydrodoisynolic acid (Z-BDDA) is a seco-steroid that has potent estrogenic as well as antioxidant activities in vitro and in vivo. We evaluated the therapeutic potential of Z-BDDA (300μg/0.1cc/100g body weight, sc) to promote the recovery of behavioral function following lateral fluid percussion injury (FPI) to the brain in male rats. Two hours subsequent to FPI, treatment with Z-BDDA began with a bolus subcutaneous (sc) injection followed by booster treatments given 24 and 48h later. Behavioral testing was initiated on the second day after FPI and results of Z-BDDA treatments were compared to treatment with vehicle only and to sham FPI surgery. Z-BDDA effectively enhanced recovery of coordinated limb movement assessed by locomotor placing performance across the duration of the study. Z-BDDA treated animals also performed better on a spatial memory task in the Morris water maze, showing improved learning curves across days of testing. Vestibulomotor function, measured by beam walk performance, appeared to improve in Z-BDDA treated animals, however these results did not reach statistical significance (p>0.05). Following cessation of the behavioral testing, all animals underwent assessments of gross neuroanatomical pathology. Cortical lesion size and cell death analysis with Fluoro-jade B failed to reveal Z-BDDA enhanced neuroprotection. These findings support our hypothesis that Z-BDDA can facilitate behavioral recovery following FPI in adult male rats although the mechanism(s) of these effects remain to be determined.

Evaluation of Degenerating Neurons After Traumatic Brain Injury: Cyromicrotomy and Staining Procedures for Frozen Sections of Rat Brains

Abstract Historically, laboratories investigating traumatic brain injury (TBI) using rats as models rely on empirical stains and immunocytochemistry for evaluating inflammation, degeneration of neurons and neuronal death. Customarily, these protocols use 20- to 60-μ frozen sections that are either stained in a “free-floating” state or mounted directly onto slides. Metachromatic stains, such as formal thionin, are desirable for these studies both for their swiftness to produce data and for their simplicity. Used primarily as markers of overall histomorphology, these stains allow for quick labeling and general evaluation of target tissues. The neuroanatomical effects of TBI are demonstrated, with varying degrees of success, with silver stains and immunocytochemistries; however, established protocols provide inconsistent data and sometimes questionable results attributable to staining inconsistencies, variable section thickness, mounting artifacts, and other technical problems. This work describes a modification of traditional staining methods and innovative section-mounting technique that provides reliable and reproducible data for the evaluation of morphological changes resulting from TBI (The J Histotechnol 30:109, 2007).