Syndrome and outcome of antibody-negative limbic encephalitis.

BACKGROUND AND PURPOSE: The aim was to report the clinical characteristics of 12 patients with limbic encephalitis (LE) who were antibody-negative after a comprehensive immunological study. METHODS: The clinical records of 163 patients with LE were reviewed. Immunohistochemistry on rat brain, cultured neurons and cell-based assays were used to identify neuronal autoantibodies. Patients were included if (i) there was adequate clinical, cerebrospinal fluid (CSF) and magnetic resonance imaging information to classify the syndrome as LE, (ii) magnetic resonance images were accessible for central review and (iii) serum and CSF were available and were confirmed negative for neuronal antibodies. RESULTS: Twelve (7%) of 163 LE patients [median age 62 years; range 40-79; 9 (75%) male] without neuronal autoantibodies were identified. The most frequent initial complaints were deficits in short-term memory leading to hospital admission in a few weeks (median time 2 weeks; range 0.5-12). In four patients the short-term memory dysfunction remained as an isolated symptom during the entire course of the disease. Seizures, drowsiness and psychiatric problems were unusual. Four patients had solid tumors (one lung, one esophagus, two metastatic cervical adenopathies of unknown primary tumor) and one chronic lymphocytic leukemia. CSF showed pleocytosis in seven (58%) with a median of 13 white blood cells/mm3 (range 9-25). Immunotherapy included corticosteroids, intravenous immunoglobulins and combinations of both drugs or with rituximab. Clinical improvement occurred in six (54%) of 11 assessable patients. CONCLUSIONS: Despite the discovery of new antibodies, 7% of LE patients remain seronegative. Antibody-negative LE is more frequent in older males and usually develops with predominant or isolated short-term memory loss. Despite the absence of antibodies, patients may have an underlying cancer and respond to immunotherapy.

Transforming growth factor-alpha (TGF-alpha) and epidermal growth factor-receptor (EGF-R) immunoreactivity in normal and pathologic brain.

Transforming growth factor alpha (TGF-alpha) and epidermal growth factor-receptor (EGF-R) immunoreactivity is observed in the majority of neurons, and in maturing astrocytes, in the developing and adult brain of humans and different species of animals. TGF-alpha and EGF-R co-localize in most neurons and maturing astrocytes, suggesting that most TGF-alpha-producing cells are EGF-R-expressing cells. TGF-alpha and EGF-R immunoreactivity decrease in damaged areas following different insults. However, EGF-R appears in reactive glia, mostly reactive astrocytes, within and surrounding the damaged areas. TGF-alpha and EGF-R immunoreactivity is found in neurons of patients affected by Alzheimer's disease and other forms of dementia, and in neurons of patients suffering from epilepsy owing to different causes, thus pointing to the conclusion that TGF-alpha does not play a significant role in these pathologies. However, EGF-R immunoreactivity occurs in reactive astrocytes and microglia in subacute but not chronic lesions in human cases. Since TGF-alpha is a membrane-anchored growth factor, which may be cleaved leading to the formation of soluble forms, and both the membrane-anchored and soluble forms have the capacity to activate the EGF-R, it is feasible that TGF-alpha in the nervous system may act upon EGF-R-containing neurons through different mechanisms. In addition to distant effects resulting from the release of soluble TGF-alpha, local effects may be produced by establishing direct cell-to-cell contacts (juxtacrine stimulation), or in cells expressing both TGF-alpha and EGF-R (autocrine stimulation).

[Serotonin syndrome: report of two cases and review of the literature]. / Síndrome serotoninérgico: presentación de dos casos y revisión de la bibliografía.

INTRODUCTION: Serotonin is a neurotransmitter synthesized from tryptophan. It is implied in the regulation of mood, cognition, sleep cycle, synthesis of cerebrospinal fluid, and other processes. Generally, it is implied in human pathology by hypofunction. However, there is a complication of unknown incidence related to treatment with drugs that increase the stimulation of 5-HT1A serotonin receptors, called serotonin syndrome (SS). Clinically, it is characterised by the presence of a triad of mental and autonomic disorders, and motor hyperactivity. This entity has not biological markers and its diagnosis could be done verifying the proposed criteria. CASE REPORTS: Two cases of SS are presented, one of them related to the combination of risperidone and sertraline, as first report in the literature. Both cases had a favourable outcome employing support measures. CONCLUSIONS: The physiopathology, the diagnosis, the differential diagnosis, and the treatment are reviewed. We emphasize the potentially high frequency of this disorder, given the growing use of serotonin activity modifying drugs, and the typically benign course of the SS once the support measures are started.

BDNF and TrkB co-localize in CA1 neurons resistant to transient forebrain ischemia in the adult gerbil.

Delayed cell death of projection cells in the CA1 area of the hippocampus is produced in the adult gerbil following 5 minutes (min) of transient forebrain ischemia. Parvalbumin-immunoreactive local-circuit neurons are resistant to the ischemic insult. Brain-Derived Neurotrophic Factor (BDNF) immunoreactivity is localized in all neurons of the CA1 area in control gerbils. However, TrkB immunoreactivity is observed in a minority of BDNF-immunoreactive neurons in the CA1 area. The number of BDNF-immunoreactive cells in CA1 is dramatically reduced in ischemic gerbils as early as 24 h after ischemia, but the number of TrkB-immunoreactive cells in the CA1 area is maintained following ischemia. Moreover, about 90% of BDNF-immunoreactive cells and about 85% of TrkB-immunoreactive cells in ischemic gerbils co-localize the calcium-binding protein parvalbumin. Finally, BDNF and TrkB are coexpressed in about 95% of CA1 neurons surviving the ischemic insult. These results indicate that a subpopulation of CA1 hippocampal neurons coexpressing TrkB, parvalbumin and BDNF is resistant to transient forebrain ischemia in the gerbil. These results also suggest that a subpopulation of CA1 hippocampal neurons in the gerbil hippocampus is endowed with a putative BDNF/TrkB autocrine regulatory loop that may be involved in both cell survival and synaptic remodeling of the damaged gerbil hippocampus following transient forebrain ischemia.

BDNF up-regulates TrkB protein and prevents the death of CA1 neurons following transient forebrain ischemia.

The neurotrophin family of growth factors, which includes Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT3) and Neurotrophin-4/5 (NT4/5) bind and activate specific tyrosine kinase (Trk) receptors to promote cell survival and growth of different cell populations. For these reasons, growing attention has been paid to the use of neurotrophins as therapeutic agents in neurodegeneration, and to the regulation of the expression of their specific receptors by the ligands. BDNF expression, as revealed by immunohistochemistry, is found in the pre-subiculum, CA1, CA3, and dentate gyrus of the hippocampus. Strong TrkB immunoreactivity is present in most CA3 neurons but only in scattered neurons of the CA1 area. Weak TrkB immunoreactivity is found in the granule cell layer of the dentate gyrus. Unilateral grafting of BDNF-transfected fibroblasts into the hippocampus resulted in a marked increase in the intensity of the immunoreaction and in the number of TrkB-immunoreactive neurons in the granule cell layer of the dentate gyrus, pre-subiculum and CA1 area in the vicinity of the graft. No similar effects were produced after the injection of control mock-transfected fibroblasts. Delayed cell death in the CA1 area was produced following 5 min of forebrain ischemia in the gerbil. The majority of living cells in the CA1 area at the fourth day were BDNF/TrkB immunoreactive. Unilateral grafting of control mock-transfected or BDNF fibroblasts two days before ischemia resulted in a moderate non-specific protection of TrkB-negative, but not TrkB-positive cells, in the CA1 area of the grafted side. This finding is in line with a vascular and glial reaction, as revealed, by immunohistochemistry using astroglial and microglial cell markers. This astroglial response was higher in the grafted side than in the contralateral side in ischemic gerbils, but no differences were seen between BDNF-producing and non-BDNF-producing grafts. However, grafting of BDNF-producing fibroblasts two days before ischemia significantly and specifically prevented nerve cells from dying in the CA1 area of the ipsilateral hippocampus. Cell survival was associated with increased TrkB immunoreactivity as the majority of living cells were TrkB immunoreactive. Thus, our results show that BDNF is able to up-regulate the expression of TrkB in control and pathological states, and that BDNF prevention of neuronal death following transient forebrain ischemia is associated with increased expression of its specific receptor.

Multiple neurotrophic signals converge in surviving CA1 neurons of the gerbil hippocampus following transient forebrain ischemia.

Delayed cell death involving the CA1 area of the hippocampus was produced following 5 minutes of transient forebrain ischemia in gerbils. Cell death mainly affected CA1 pyramidal neurons, whereas parvalbumin-immunoreactive (parv-ir) cells were spared. Synaptophysin immunoreactivity was observed in the strata oriens and radiatum of CA1 for months, although immunoreactivity decreased in gerbils surviving 1 year post-ischemia. Golgi studies disclosed a few pyramidal neurons with dendrites, variably covered with dendritic spines, in the CA1 area of 1-year surviving gerbils. In the normal gerbil, the majority of CA1 neurons expressed brain-derived neurotrophic factor (BDNF), tyrosine protein kinase C (TrkC), fibroblast growth factor receptor 1 (Flg), transforming growth factor-alpha (TGF-alpha), and epidermal growth factor-receptor (EGF-R), but only a minority of cells were tyrosine protein kinase B (TrkB)-immunoreactive. Marked reduction in the number of BDNF-, TrkC-, Flg-, TGF-alpha-, and EGF-R-ir cells was observed in CA1 from 24 hours to 1 year after ischemia. In contrast, TrkB-ir cells survived the ischemic insult. Double-labeling immunohistochemistry disclosed that about 90% of surviving BDNF-ir and 85% of TrkB-ir neurons co-localized parvalbumin in the CA1 area. In control gerbils, only about 5% of BDNF-ir cells in CA1 co-expressed TrkB. However, TrkB co-localized in about 95% of surviving BDNF-ir neurons in CA1 in ischemic gerbils. In addition, parvalbumin was co-expressed in about 90% of TrkC-, 95% Flg-, and 85% EGF-R-ir surviving neurons in the stratum pyramidale of CA1. Finally, basic fibroblast growth factor (bFGF) was expressed by reactive astrocytes from day 4 onwards. These data show that the subpopulation of TrkB-/parv-ir neurons in CA1 survive the ischemic episode and that multiple neurotrophic signals converge in surviving neurons of the gerbil hippocampus following transient forebrain ischemia.

Transient forebrain ischemia in the adult gerbil is associated with a complex c-Jun response.

C-Jun expression in the hippocampus of gerbils subjected to 5 min of transient forebrain ischemia was examined with immunohistochemistry and western blotting using two c-Jun antibodies raised against two different amino acid sequences. Both c-Jun antibodies showed increased immunoreactivity at 6 and 12 h postischemia in the stratum pyramidale of CA3 and granule cell layer of the dentate gyrus. No immunostaining was detected in CA1 up to the 7th day. Western blots showed increased c-Jun immunoreactivity at 6 and 12 h. However, the antibody c-Jun (AB-1) detected a single band at about p39 in normal and post-ischemic states, whereas the antibody c-Jun/AP-1 (N) recognized a band at about p39 in normal and post-ischemic gerbils, and a p62 phosphorylated double-band at 6 and 12 h following ischemia. In addition, increased c-Jun N-terminal kinase-1 (JNK-1) expression was observed on western blots at 6 and 12 h postischemia. These results suggest that different c-Jun-related responses, some of which probably indicate post-translational changes of the c-Jun protein, occur in the hippocampus of the gerbil following transient forebrain ischemia.

Strong c-Jun/AP-1 immunoreactivity is restricted to apoptotic cells following intracerebral ibotenic acid injection in developing rats.

Strong c-Jun immunoreactivity, as revealed with the antibody c-Jun/activator protein 1 (AP-1) which is raised against the amino acids 91-105 mapping with the amino terminal domain of mouse c-Jun p39, is observed in apoptotic cells, but not in necrotic cells, following intracerebral injection of ibotenic acid in the developing rat brain processed for immunohistochemistry. Immunostaining occurs in the cytoplasm and dendrites, thus suggesting impaired nuclear translocation of c-Jun in apoptotic cells. Western blotting of total brain homogenates, using the same antibody, shows a band at p39 which is more marked in treated animals than in age-matched controls. In addition, increased c-Jun N-terminal kinase 1 (JNK-1) expression, as revealed on Western blots, is found in rats treated with ibotenic acid when compared with controls. In contrast, apoptotic cells are not stained with antibodies to Jun B and Jun D. These results give further support to previous studies showing strong c-Jun expression in apoptotic cells at determinate stages of development, and emphasize that intracellular distribution of c-Jun, possible post-translational modifications of c-Jun due to phosphorylation at specific transactivation sites, and lack of associated Jun B and Jun D expression may differentiate the Jun response in apoptotic cells from other forms of cellular response involving c-Jun which are not associated with cell death.

bFGF and FGFR-3 immunoreactivity in the rat brain following systemic kainic acid administration at convulsant doses: localization of bFGF and FGFR-3 in reactive astrocytes, and FGFR-3 in reactive microglia.

Strong bFGF immunoreactivity was observed in reactive astrocytes, as shown by double-labeling immunohistochemistry of bFGF and GFAP, from days 7 up to 30 (last time point examined) following kainic acid (KA) injection at convulsant doses in the adult rat. bFGF was not found in OX-42-positive reactive microglia. A few reactive glia co-localized FGFR-3 and GFAP, whereas the majority of cells expressing FGFR-3 were OX-42-immunoreactive. This was further supported by the observation that only approximately 10% of reactive glia co-localized bFGF and FGFR-3. These results show that reactive astrocytes are a major source of bFGF during the subacute stages of tissue damage following KA injection and that reactive astrocytes and, most particularly, reactive microglia are putative targets of bFGF through FGFR-3.

CREB-1 and CREB-2 immunoreactivity in the rat brain.

This study is focused to learn about the cellular localization of transcription factors binding to the cAMP response element-CREBs-in the brain of normal rats and in animals subjected to excitotoxic cell damage. For this purpose, CREB-1 and CREB-2 immunoreactivity is examined in the developing and adult rat brain under physiological conditions, and following systemic kainic acid (KA) injection at convulsant doses in the adult, as a validated experimental model of excitotoxic injury. CREB-1 immunoreactivity is constitutively expressed in periventricular glia and Bergmann glia, and appears in reactive astrocytes following KA-induced excitotoxic cell damage. In contrast, CREB-2 is constitutively expressed in all neurons of the cerebrum, cerebellum and brain stem in the developing and adult brain. CREB-2 immunoreactivity is not increased following KA excitotoxic cell damage. These results demonstrate that CREB-1 and CREB-2 in the brain of the rat are localized in separate cellular compartments and that their expression is differentially regulated in pathologic states.

Amoeboid microglial response following X-ray-induced apoptosis in the neonatal rat brain.

The phagocytic response following X-ray-induced apoptosis in the neonatal rat brain was examined by immunohistochemistry with the antibodies OX-6 and OX-42 which recognize MHC class II antigens and the CR3 complement receptor, respectively. Few OX-6-immunoreactive cells were observed in control rats, and in rats irradiated at postnatal day 2 and examined during the first 2 postnatal weeks. However, a transient increase in the number of OX-42-immunoreactive amoeboid microglia, containing large numbers of apoptotic cells, occurred at 6, 24 and 48 h after irradiation when compared with age-matched controls. These results show that X-ray-induced apoptosis promotes a short-lasting phagocytic response.

L-Deprenyl does not reduce brain damage in global forebrain ischemia in adult gerbils (Meriones ungiculatus).

Delayed neuronal death is produced at about the 4th day following global forebrain ischemia. This study investigates whether L-deprenyl, an irreversible and selective MAO-B inhibitor, reduces brain damage following global forebrain ischemia in adult gerbils. For this purpose, global forebrain ischemia was induced in adult gerbils by occlusion for 5 min of both common carotid arteries. L-Deprenyl, 10 mg/kg weight in saline (10 mg/ml) i.p., was administered 1 h after or 2 h before occlusion, followed by daily administration for 4 days. Treated animals were processed in parallel with ischemic animals receiving saline alone, and with sham-operated controls. Counts of viable neurons were made in the pyramidal cell layer of the CA1 region of the hippocampus at the 4th day after the ischemic episode. The number of viable neurons in the pyramidal cell layer of CA1 was similar in animals treated with L-deprenyl or saline alone (Mann-Whitney U-test, alpha=0.05 two-tailed). The present results show that L-deprenyl does not prevent neuronal cell death following global forebrain ischemia in the adult gerbil when the administration of the drug is started shortly after or shortly before the ischemic episode.

[Cerebrovascular pathology: inpatient study in a community hospital. A prospective study]. / Patología cerebrovascular intrahospitalaria en el ámbito comarcal: un estudio prospectivo.

We present the preliminary results of six months cerebrovascular pathology follow-up carried out at the Sant Boi Local Hospital as from when a Neurological Unit was set up within the Internal Medecine Service. For this purpose we drew up a prospective study based on questionnaires. A total of 69 neurological patients (2.45% of all those hospitalized) were studied, 62.3% of whom suffered acute stroke. Diagnostic and therapeutic activities were analyzed with regards to the afore mentioned pathology, as well as various data of an epidermiological nature. Lastly we present the experimental multidisciplinary approach to acute stroke as performed during hospitalization by the Internal Medecine Service, Convalescence Unit and an Interdepartmental Socio-Sanitary Functional Unit.

[Cost effectiveness analysis of the diagnosis of carpal tunnel syndrome using electrophysiological studies]. / Análisis coste-efectividad del diagnóstico del síndrome del túnel carpiano mediante estudio electrofisiológico.

INTRODUCTION: The high demand for electrophysiological studies (EP studies) in paucisymptomatic patients with suspected carpal tunnel syndrome (CTS) creates overloads in neurological examination departments. Most of these requests in the referenced section of our hospital come from primary health care, however it also comes from other specialists. Many EP Studies for CTS are normal or have minimal alterations so that no change in the therapeutic attitude is required. Thus, it is not clear whether EP studies are cost-effective for suspected CTS without clinical evidence of axonal degeneration. METHODS: A decision-making and economic evaluation model was made to compare three strategies: option A (EP Studies for all patients with suspected CTS), option B (prior selection by neurologist and EP studies only if axonal CTS was suspected) and option C (prior selection by neurologist and EP studies only if CTS, axonal or otherwise was suspected). The study was conducted over a two month period on patients referred to the neurology department with suspected CTS. EP studies were considered to be the proof that established the final diagnosis. The consequences were determined in terms of correct diagnoses, cost and cost/effectiveness ratio for each strategy. A total number of 188 studies were performed, option C being the most cost-effective, but also the most expensive. CONCLUSIONS: When there is neurological screening, the cost-effectiveness ratio is better but it is also more expensive. It is recommended to favor option A with intermediate cost and an acceptable cost-effectiveness ratio. However, this also generates great pressure on bud- 45 gets and care facilities which means that CTS diagnosis and criteria for referring patients to the neurological examination departments must be improved.

MR assessment of the repaired rotator cuff: prevalence, size, location, and clinical relevance of tendon rerupture.

The objectives of this study were to use magnetic resonance (MR) imaging to evaluate the prevalence, size, location, and clinical relevance of tendon rerupture following complete repair of full-thickness rotator cuff tear (RCT). A total of 78 surgically proven full-thickness rotator cuff tears in 74 patients were retrospectively included in the study. Clinical assessment was performed using the University of California at Los Angeles score. Postoperative MR imaging was evaluated to determine prevalence, size, and location of tendon rerupture. At a mean 48.4 months' follow-up, 62 shoulders (79.5%) had favorable outcomes and 45 shoulders (57.6%) showed rerupture on MR imaging studies. Reruptures were significantly more prevalent among patients with intermediate-to-bad outcomes (81.3%), with surgically demonstrated two-tendon tears (78.9%) or three-tendon tears (100%), and with preoperative fatty degeneration of the supraspinatus muscle greater than 1 (91.6%). Reruptures were also significantly larger in those subgroups. Complete repair of RCT of all sizes may have favorable outcomes in a significant proportion of patients in spite of a high prevalence of reruptures. Preoperative tear size and degree of muscle fatty degeneration influence the prevalence and rerupture size. After repair of supraspinatus tears, reruptures tend to invade the posterior aspect of the tendon.

Kainic acid-induced excitotoxicity is associated with a complex c-Fos and c-Jun response which does not preclude either cell death or survival.

c-fos and c-jun mRNA induction and c-Fos and c-Jun protein expression were examined in the brains of adult rats subjected to systemic kainic acid (KA) injection at convulsant doses. Induction of c-fos and c-jun mRNA, as seen with in situ hybridization, occurred in the piriform and entorhinal cortices, neocortex, amygdala, hippocampus, dentate gyrus, and discrete thalamic nuclei. This was followed by c-Fos protein expression, as revealed with immunohistochemistry, in the same regions. However, the distribution of c-Jun protein expression differed depending on the antibody used. The distribution of cells immunostained with the antibody c-Jun (AB-1) was similar to that of c-jun mRNA, but the distribution of cells immunostained with the antibody c-Jun/AP1 (N) was restricted to a few neurons in the pyramidal cell layer of CA1 and CA3, layer II of the piriform and entorhinal cortices, basal amygdala, and discrete thalamic nuclei. Although the regional distribution of c-Fos- and c-Jun-immunoreactive cells in the hippocampus, layer II of the entorhinal and piriform cortices, basal amygdala, and discrete thalamic nuclei matched the distribution of cells committed to dying, c-Fos- and c-Jun-immunoreactive cells in the neocortex and dentate gyrus survived. Therefore, the present data show that c-fos and c-jun are not predictors of either cell death or survival, but rather, markers of cells sensitive to KA excitotoxicity. Western blots to c-Fos showed a double band at p62 in samples containing the hippocampus and entorhinal and piriform cortices (hip samples) and in samples containing the neocortex (cortex samples). The upper band was abolished following preincubation of the samples with alkaline phosphatase, thus suggesting c-Fos phosphorylation. Western blots to c-Jun (AB-1) showed a single band at about p39 in hip and cortex. However, Western blots to c-Jun/AP1 (N) identified two bands. One band at about p39 was seen in control rats and the cortex of KA-treated rats. Another band at p26 was observed only in hip samples of KA-treated rats. In addition, decreased c-Jun N-terminal kinase 1 (JNK-1) expression, as revealed on Western blots, was coincidental with the appearance of the p26 c-Jun-immunoreactive band in KA-treated rats. These results show that c-Fos and different Jun-related antigens are expressed following KA excitotoxicity, and that posttranslational modifications involving phosphorylation of c-Fos and Jun(s) may occur following KA injection. These results also stress the necessity of examining the composition of Fos and Jun-related antigens and the metabolic state of Fos and Jun(s) in different experimental models of nervous system injury.

TrkA immunoreactivity in reactive astrocytes in human neurodegenerative diseases and colchicine-treated rats.

It has been shown that nerve growth factor (NGF) administration is capable of curbing tissue damage in several neurodegenerative disorders. As a first step to learning about the possible functional role of NGF in the astroglial response during neurodegeneration, we have analyzed the expression of the functional receptor for NGF, TrkA, in human neurodegenerative diseases which are accompanied by reactive astrocytosis, as well as in human astrocytomas. We have compared these results with those observed in reactive astrocytes following colchicine-induced cellular damage to adult rats. In the human brain, strong TrkA immunoreactivity is observed in reactive astrocytes in a number of unrelated diseases, including Alzheimer's disease, Huntington's disease, progressive supranuclear palsy, multiple sclerosis, Creutzfeldt-Jakob disease, multifocal leukoencephalopathy and residual hypoxic encephalopathy. Neoplastic astrocytes in grade II and III astrocytomas display strong TrkA immunoreactivity. In the rat brain, reactive astrocytes following mechanical needle injury and colchicine administration show strong TrkA immunoreactivity. The presence of TrkA receptors in reactive astrocytes from different human neurodegenerative diseases and experimentally induced models in rats, and in neoplastic astrocytes suggests that NGF may participate in the astroglial response to different types of injury and neoplastic proliferation. Since astroglial cells are capable of producing NGF, it is plausible that this neurotrophin may function as an autocrine or paracrine factor in TrkA-expressing reactive and neoplastic glial cells.

Increase in SNAP-25 immunoreactivity in the mossy fibers following transient forebrain ischemia in the gerbil.

SNAP-25 (a synaptosomal-associated protein of 25 kDa) has been shown to be involved both in synaptic vesicle exocytosis and in axonal outgrowth. In the present study, we investigated the changes in SNAP-25 immunoreactivity in the hippocampus of the Mongolian gerbil (Meriones unguiculatus) at different time points after transient forebrain ischemia insult. In parallel, immunostaining for GAP-43, a protein involved in axonal outgrowth, and for syntaxin-1 (stx1A and stx1B), another protein implicated in neurotransmitter release, was also analyzed. The animals were subjected to 2.5 or 5 min of transient forebrain ischemia through bilateral common carotid occlusion, and examined at different intervals after ischemia. SNAP-25 immunoreactivity was increased in the mossy fiber layer as early as 2 days after 5 min of ischemia. Increased SNAP-25 immunoreactivity in mossy fibers was also detected at days 4 and 7 after ischemia. On day 15, SNAP-25 staining was similar to that observed in control non-ischemic animals. In contrast, no changes in GAP-43 and syntaxin-1 immunoreactivity were observed in the mossy fiber layer following 5 min of ischemia. No modifications in SNAP-25, syntaxin-1 or GAP-43 immunoreactivity were observed following 2.5 min of ischemia, the longest period for which no neuronal damage is observed. These results provide evidence of a specific involvement of SNAP-25 in the reactive changes associated with transient forebrain ischemia.

Bcl-2, Bax and Bcl-x expression following hypoxia-ischemia in the infant rat brain.

Severe hypoxic-ischemic cerebral damage was produced in 8-day-old rats following permanent bilateral carotid artery occlusion and 15 min of ischemia. Cellular damage consisted of early necrosis and appearance of cells with apoptotic-like morphology (karyorrhectic cells) and cells with granular chromatin degeneration in the cerebral cortex, hippocampus, thalamus, stratum and amygdala. Expression of Bcl-2, Bax and Bcl-x was examined in control and hypoxic-ischemic rats using immunohistochemistry and Western blotting. Members of the Bcl-2 family were expressed in the vast majority of, if not all, neurons in control pups. Bcl-2, Bax and Bcl-x immunoreactivity decreased in necrotic cells, but about 60% of cells with apoptotic-like morphology and cells with granular chromatin degeneration were stained with antibodies to Bcl-2, Bax or Bcl-x. Although the total number of stained cells decreased with time, recruitment of cells with apoptotic morphology and cells with granular chromatin degeneration was still found up to 48 h. Western blots showed a band at about p28 and p21, respectively for Bcl-2 and Bax in control and hypoxic-ischemic pups at 6, 12 and 24 h. Two bands at about p37 and p30, representing Bcl-xL and Bcl-xS, respectively, were found in samples stained with antibodies to Bcl-x. No gross changes in the intensity of these bands were observed in ischemic pups at 6, 12 and 24 h, except for a slight decrease in Bcl-2 at 24 h, and a slight and inconstant increase of the putative Bcl-xS at 12 h. These results suggest that Bcl-2, Bax, Bcl-xL and Bcl-xS do not play a leading role in the fate of damaged nerve cells following a severe hypoxic-ischemic insult of the developing brain.