Graft-induced behavioral recovery from subcallosal septohippocampal damage in rats depends on maturity stage of donor tissue.

Long-Evans female rats sustained electrolytic lesions of the fimbria and the dorsal fornix and, 10-14 days later, received intrahippocampal suspension grafts of septal-diagonal band tissue from either 14-day-old (Group S14, n = 8) or 16-day-old fetuses (Group S16, n = 10), or of parietal cortex from 16-day-old fetuses (Group Cx, n = 10). Sham-operated (Group S, n = 10) and lesion-only (Group Fifo, n = 21) rats served as non-grafted controls. Spontaneous alternation was assessed in a T-maze at three weeks and two months post-grafting. Home cage and open field activity as well as radial maze learning were assessed from two months post-grafting onwards. Fimbria-fornix lesions induced lasting hyperactivity in both the open field and the home cage, impaired radial maze learning and transiently reduced spontaneous alternation rates. Neither type of graft significantly affected home cage activity. Septal-diagonal band grafts improved open field habituation (within trial decline of ambulatory activity) and radial maze learning; the former was observed only in S16 rats, whereas the latter was observed only in S14 rats. Acetylcholinesterase histochemistry revealed an initial lesion-induced depletion of hippocampal acetylcholinesterase (eight days post-surgery) which was no longer observed at the end of the experiment. Acetylcholinesterase positivity was similar in S14 and S16 grafts, which also contained many choline acetyltransferase-positive neurons. Cortical grafts were found to be almost devoid of acetylcholinesterase positivity and no well-stained choline acetyltransferase-positive neurons could be identified. Septal-diagonal band grafts from 14-day-old fetuses and cortical grafts contained more parvalbumin-positive neurons than septal-diagonal band grafts provided by 16-day-old fetuses. These results suggest that grafts rich in cholinergic neurons may promote behavioral recovery from fimbria-fornix lesion-induced deficits. However, such a recovery may concern different behavioral deficits as a function of the age of the implanted tissue, suggesting that the maturity stage of the donor may critically influence the functional expression in the lesioned recipient. Also, such a recovery does not appear to be related solely to cholinergic hippocampal (re)innervation and might depend on the presence, not only of cholinergic neurons, but also of non-cholinergic neuronal populations, such as parvalbumin-positive (probably GABAergic) neurons.

Neuroprotective effects of HU-211 on brain damage resulting from soman-induced seizures.

Neuroprotective effects of HU-211 (dexanabinol), a synthetic nonpsychotropic analog of tetrahydrocannabinol, on brain damage resulting from soman-induced seizures were examined in male Sprague-Dawley rats challenged with 1.6 LD50 soman. At 5 or 40 min after onset of seizures, the rats were given an intraperitoneal injection of 25 mg/kg HU-211. All rats that received soman showed electrocorticographic (ECoG) evidence of sustained seizures and status epilepticus for 4-6 hr. HU-211 had no effect on either the strength or duration of seizure activity. Administration of HU-211 at 5 min after seizure onset reduced median lesion volume 86% (as assessed by microtubule-associated protein 2 (MAP2)-negative staining), and when administered 40 min post-onset, the reduction in necrosis was 81.5% despite the presence of continuous seizures for 4-5 hr. These observations were corroborated by hemotoxylin and eosin (H&E) histopathological assessment that showed a significant reduction in piriform cortical neuronal damage in HU-211-treated animals. It is concluded that HU-211 provides considerable neuroprotection against brain damage produced by soman-induced seizures.

Microtubule-associated protein 2 (MAP-2): a sensitive marker of seizure-related brain damage.

We have assessed the efficacy of MAP-2 immunohistochemistry as a marker of seizure-related brain damage and its suitability for quantitation of the damage using densitometric and morphometric image analysis. Seizures were produced in rats by administration of 1.5 LD50 soman, an irreversible AChE inhibitor. Our results demonstrate that neuronal damage, assessed using hematoxylin and eosin, and cresyl violet staining, was colocalized on adjacent serial sections with clearly demarcated reductions in MAP-2 staining. The most severely damaged brain regions were devoid of MAP-2 staining. Reductions in MAP-2 immunostaining were found to be exceptionally well suited for quantitation using densitometric and morphometric image analysis. This study represents the first demonstration of seizure-induced excitotoxic alterations in MAP-2.

Injections of fluid or septal cell suspension grafts into the dentate gyrus of rats induce granule cell degeneration.

This study was originally aimed at investigating the effects of intragyral cell suspension grafts which had been enriched in basic fibroblast growth factor (bFGF) before being implanted into the rat hippocampus denervated by aspiration of the septohippocampal pathways. Whether treated with vehicle alone, vehicle + bFGF, cell suspension with or without bFGF, and irrespective of the surgical treatment (sham-operation, lesions or lesions + grafts), we unexpectedly found approximately 80% of the rats to show morphological alterations in the dentate gyrus (20 weeks post-grafting). These alterations consisted of loss of a part of the granule cells; this loss was most often located in the dorsal leaf of the dentate gyrus. Also, in the close vicinity of the degeneration area, we found severe shrinkage of the molecular layer and disappearance of the typical laminae pattern of acetylcholinesterase distribution. These observations confirm previous findings which showed that fluid injections into the dentate gyrus, a widely used technique for intracerebral administration of drugs, trophic factors or neural grafts, may induce undesirable granule cell necrosis.

Cytophotometric assessment of granulosa cell protein and nucleic acid levels during the estrous cycle in rats treated with T-2 toxin.

Female Sprague-Dawley rats (160-180g.) with normal estrous cyclicity established by vaginal smears, were injected intraperitoneally with 0.45 mg/kg (low dose) or 0.68 mg/kg (high dose) of T-2 toxin, a trichothecene with potent protein inhibitory abilities. Control animals were injected with only the 100% ethanol vehicle. All animals were decapitated at 8 hours post-exposure, and their ovaries removed and processed for paraffin sectioning. Coomassie, Feulgen, and azure B/DNase staining procedures were used to show granulosa cell protein levels, F-DNA stainability, and basophilia/RNA levels, respectively. Quantification of these parameters was accomplished using scanning-integrating microdensitometry. T-2 toxin treatment groups had granulosa cell protein levels significantly lower than those of the control animals. However, rats exposed to the lower dose of T-2 toxin generally showed a more marked suppression of protein levels than the high dose group, regardless of the stage of the estrous cycle. In addition, rats that received lower doses of T-2 toxin had impaired translation and template activity in response to injury, when compared with the rats in the high dose group. These results are attributed to the lesser degree of circulatory impairment in the low T-2 toxin dosage group, which allows a higher amount of T-2 toxin to interact with the cells.

Cytophotometric analysis of T-2 toxin induced alterations in chromatin condensation and neuronal nuclear volume of rat supraoptic-magnocellular neurons.

Quantitative cytophotometry and ocular filar micrometry were used to monitor T-2 toxin induced alterations in chromatin and neuronal nuclear volume in supraoptic-magnocellular neurons of rat hypo-thalami. Thirty male Sprague-Dawley rats (200-220g) were given a single i.p. injection of T-2 toxin (0.5, 0.75, 1.0 and 1.5 X LD50), a trichothecene mycotoxin; rats were decapitated 8 hours post-dosing. After stoichiometric Feulgen-DNA staining of brain sections, scanning-integrating microdensitometry was used to quantify changes in the susceptibility of chromatin to Feulgen acid hydrolysis. Changes in neuronal nuclear volumes were also determined histometrically. Within the magnocellular neurons of the supraoptic nuclei, significant reductions in F-DNA reactivity were observed in the 0.5, 0.75, and 1.0 X LD50 groups (i.e. 3.7%, 4.4% and 2.5%, respectively); however, rats receiving 1.5 X LD50 T-2 toxin showed no difference in F-DNA reactivity compared to controls. In addition, ocular filar micrometry demonstrated increased neuronal nuclear volumes in all groups receiving T-2 toxin, and following an inverse trend to that seen with F-DNA stainability. Additional observations included pronounced polydipsia, polyphagia and horripilation in the experimental groups, independent of the dosages employed; these changes were evident within 1 hour post-injection. It is postulated that the T-2 toxin induced reduction in the susceptibility of chromatin to Feulgen acid hydrolysis and concomitant increases in neuronal nuclear volumes represent an early indication of impaired metabolic activity. Since these neurons are important sites of vasopressin (antidiuretic hormone) synthesis, these data suggest an impaired osmoregulatory ability. The pronounced polydipsia which occurred shortly after intoxication is further evidence of this impairment. Although these findings do not provide insight relating to the mechanism of osmoregulatory disruption, it is evident that an impaired ability to osmoregulate is among the earliest indications of acute T-2 toxin mycotoxicosis.

Cytophotometric assessment of T-2 toxin induced alterations in azure B-RNA and Coomassie-protein in supraoptic-magnocellular neurons of rat hypothalami.

Quantitative cytophotometry was used to monitor T-2 toxin-induced alterations in azure B-RNA and Coomassie-total cell protein in supraoptic-magnocellular neurons of rat hypothalami. Thirty male Sprague-Dawley rats (200-220g) were given a single i.p. injection of T-2 toxin (0.5, 0.75, 1.00 and 1.50 x LD50), a trichothecene mycotoxin; rats were decapitated 8 hours post-dosing. After stoichiometric azure B-RNA and Coomassie-protein staining of brain sections, scanning-integrating microdensitometry was used to quantify toxin-induced alterations in these well established indices of neuronal toxicity. Within the magnocellular neurons of the supraoptic nuclei, significant reductions in azure B-RNA reactivity were observed in the 0.75, 1.00 and 1.50 x LD50 groups (i.e. 11%, 13% and 8%, respectively); no differences in RNA levels were observed between controls and the 0.50 x LD50 group. In addition, a decrease in Coomassie-total cell protein was seen in animals receiving 0.50, 0.75 and 1.50 x LD50 T-2 toxin (i.e. 33%, 21% and 12%, respectively); however, toxin administration did not alter protein levels in the 1.00 x LD50 group. Furthermore, a dose-dependent decrease in systolic blood pressure was observed at 8 hr. post-injections (i.e., approximately -39%, -52%, -66% and -64% for the 0.50, 0.75, 1.00 and 1.50 x LD50 groups, respectively). Additional observations include pronounced polydipsia, ascites, abdominal and subdural hemorrhage, and horripilation (piloerection) in experimental groups. It is postulated that the T-2 toxin-induced reductions in azure B-RNA and Coomassie-protein represent an early indication of impaired metabolic activity. Since these neurons are important sites of vasopressin (antidiuretic hormone) synthesis, these data suggest an impaired osmoregulatory ability. The pronounced polydipsia which occurred shortly after intoxication is further evidence of this impairment. Although these findings do not provide insight relating to the mechanism of osmoregulatory disruption, it is advanced that the supraoptic-magnocellular compartment represents an important site in T-2 toxin mycotoxicosis. Moreover, these findings support previous claims that T-2 toxin intoxication may critically impair the vasopressinergic response to toxin-induced cardiovascular collapse.

Mesenteric mast cell degranulation in acute T-2 toxin poisoning.

T-2 toxin-induced alterations in rat mesenteric mast cell granulation were measured by cytophotometric analyses of the metachromatic reaction of mast cell granules with azure B. Hypogranulation (diminution of metachromatic material) was observed 8 h following injections of T-2 toxin (0.5-1.5 LD50, i.p.). These data suggest that mast cell activation occurs during acute T-2 intoxication and raise the possibility that mast cell mediators may contribute to toxin-induced cardiovascular collapse.

Changes in HS mouse erythrocyte mass and area with respect to donor age.

Interferometric photographs of blood smears from heterogeneous stock (HS) mice were analyzed for age-related changes in erythrocyte mass using quantitative cytophotometry. Blood smears were obtained from 96 mice (half male/half female) sacrificed at 172, 272, 374, 482, 581, and 664 days of age as part of a larger biomarkers of aging study. Blood smears from each animal were photographed with a Leitz Mach-Zehnder interferometer, and the erythrocyte images on these negatives were measured for dry mass using a Vickers M85a microdensitometer. Cell area measurements were made from a video-microscope image traced with a computer digitizer. Results indicated that animals from middle-age groups (272, 374, and 482 days) have higher red blood cell mass than animals at either young (172 days) or older (581 and 664 days) age groups. Mass changes with respect to age followed similar trends when data were further examined with regard to sex of the animals. Erythrocyte area measurements showed a general age-related decrease in cell size.

Cytophotometric assessment of Feulgen-DNA and coomassie-total cell protein in adrenocortical fasciculata cells of noise-exposed Wistar rats.

The present investigation was undertaken to examine possible noise-induced alterations in adrenal fasciculata cell (AFC) metabolism, and also to determine if the magnitude of these changes differs in male versus female rats. Wistar rats approximately 3 months old were exposed to intense noise for 60 min (100 dB, re 2 x 10(-5) N(m2)-1, 350-20,000 Hz); control rats were housed under identical conditions, at an ambient noise level of 40-60 dB. Adrenal fasciculata cells (AFC) from each animal were examined for noise-induced alterations in Feulgen-DNA reactivity (as an indicator of chromatin template activity) and Coomassie-total cell protein levels using scanning-integrating cytophotometry. The results provide evidence that intense noise elicited a marked AFC metabolic enhancement in both male and female rats; the degree of this enhancement was more pronounced in males. This disparity may be due to pre-existing differences in male versus female AFC enzymatic capability and subsequent responsiveness to noise-induced activation.

Effects of soman-induced convulsions on phosphoinositide metabolism.

Turnover of [3H]phosphoinositides (PI) was examined in brain slices from the hippocampus of rats undergoing soman-induced seizure activity. Hydrolysis of PI was determined by measuring the accumulation of [3H]inositol-1-phosphate (IP1). Incubation of hippocampal slices in the presence of carbachol or norepinephrine (NE) increased PI hydrolysis. Stimulated hydrolysis by NE, but not carbachol was significantly reduced in slices from soman-challenged rats undergoing convulsive activity. NE-stimulated PI hydrolysis was not reduced in slices from animals exposed to soman that did not exhibit convulsive activity. In rats surviving for 24 h, the response to NE was not different from control rats. In control slices, NE-stimulated hydrolysis of PI was potentiated by GABA. No potentiation by GABA was seen in slices from animals undergoing seizures. Uptake and incorporation of myo-[2-3H]inositol into phospholipids was reduced in slices from rats undergoing convulsions. Reduced IP1 production appeared to be owing, in part, to decreased synthesis of inositol lipids. These observations suggest that during soman-induced seizure activity, there is an apparent decrease in the response of the PI second messenger system to NE stimulation, and that this may contribute to the severity and duration of convulsions and brain damage resulting from exposure to soman and other anticholinesterase compounds.

GM1 monosialoganglioside pretreatment protects against soman-induced seizure-related brain damage.

The effects of GM1 monosialoganglioside pretreatment on brain damage resulting from soman-induced seizure activity were examined in this study. Male Sprague-Dawley rats were infused with GM1 via an osmotic minipump connected through a permanent cannula implanted intracerebroventricularly and challenged with soman (83 micrograms/kg, i.e., 1.25 x LD50) 4 d after initiation of GM1 infusion. Electrocorticographic recordings were monitored via indwelling cortical electrodes. Twenty-seven hours after soman administration, anesthetized rats were euthanized via transcardial perfusion with buffered paraformaldehyde. Brains were processed for hematoxylin and eosin (H&E), cresyl violet (CV), and acetylcholinesterase (AChE) histochemistry, and glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2) immunohistochemistry. All soman-challenged rats not infused with GM1 (n = 14) developed status epilepticus (SE).