Humberto Milani

Vitamin E improves learning performance and changes the expression of nitric oxide-producing neurons in the brains of diabetic rats

We investigated the effects of chronic administration of vitamin E on nitric oxide (NO)-producing neurons in the brains of streptozotocin (STZ)-induced diabetic rats using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. We further evaluated the effects of diabetes and vitamin E treatment on experimental anxiety and memory processes using the elevated plus maze (EPM) Trial 1/2 protocol. Wistar rats were divided into four groups: normoglycemics (N), normoglycemics treated with vitamin E (NVE), diabetics (D), and diabetics treated with vitamin E (DVE). Diabetes mellitus was induced by a single intraperitoneal injection of STZ (35mg/kg). Vitamin E (100mg/kg) or vehicle was administered orally by gavage (1ml/kg) once each day for 7 weeks. After behavioral testing, the dentate gyrus of the hippocampus (DG), striatum, paraventricular nucleus of the hypothalamus (PVN), supraoptic nucleus (SON), and dorsolateral periaqueductal grey (DLPAG) were analyzed for NADPH-d histochemistry. STZ-induced diabetic rats exhibited decreased locomotor activity and cognitive impairment compared with normoglycemic controls. The number of NADPH-d-positive neurons was increased in the DG, striatum, and DLPAG of diabetic rats. An increase in soma area was detected in all structures analyzed (DG, striatum, PVN, SON, and DLPAG) of STZ-induced diabetic animals. The present study showed that chronic administration of vitamin E ameliorates memory in STZ-induced diabetic rats and revealed that NOS-producing neurons have an increased soma area which can be restored, at least partially, by vitamin E treatment. These results suggest the potential use of vitamin E as an adjuvant therapy for the prevention and treatment of diabetic conditions.

A novel version of the 8-arm radial maze: effects of cerebral ischemia on learning and memory.

A novel version of the 8-arm radial maze task was developed to quantify spatial learning and memory in rats subjected to transient cerebral ischemia (TCI) using the 4-VO model. This maze uses the rats natural behavior of avoiding open, illuminated areas, and preference for a darkened, enclosed shelter. Ischemic rats were required to escape from the central area into the darkened goal box. Ischemia was induced before or after training to examine its influence on acquisition and retention of cognition, respectively. During the acquisition test, latency of ischemic rats to find the goal box, and working memory performance were significantly impaired (P < 0.005-0.001). The performance for retention of cognition was also disrupted by ischemia (P < 0.05-0.01). There was no correlation between the degree of CA1 pyramidal cell loss and behavioral deficits. The present data reveal that the aversive version of the 8-arm radial maze is sensitive to the cognitive effects of ischemia. Since it excludes the need for food deprivation or immersion of the animal in water, the method should provide a sensitive and more practical behavioral test with which to evaluate the effects of ischemic brain damage on cognition.

Influence of single and repeated cannabidiol administration on emotional behavior and markers of cell proliferation and neurogenesis in non-stressed mice

Therapeutic effects of antidepressants and atypical antipsychotics may arise partially from their ability to stimulate neurogenesis. Cannabidiol (CBD), a phytocannabinoid present in Cannabis sativa, presents anxiolytic- and antipsychotic-like effects in preclinical and clinical settings. Anxiolytic-like effects of repeated CBD were shown in chronically stressed animals and these effects were parallel with increased hippocampal neurogenesis. However, antidepressant-like effects of repeated CBD administration in non-stressed animals have been scarcely reported. Here we investigated the behavioral consequences of single or repeated CBD administration in non-stressed animals. We also determined the effects of CBD on cell proliferation and neurogenesis in the dentate gyrus (DG) and subventricular zone (SVZ). Single CBD 3mg/kg administration resulted in anxiolytic-like effect in mice submitted to the elevated plus maze (EPM). In the tail suspension test (TST), single or repeated CBD administration reduced immobility time, an effect that was comparable to those of imipramine (20 mg/kg). Moreover, repeated CBD administration at a lower dose (3 mg/kg) increased cell proliferation and neurogenesis, as seen by an increased number of Ki-67-, BrdU- and doublecortin (DCX)-positive cells in both in DG and SVZ. Despite its antidepressant-like effects in the TST, repeated CBD administration at a higher dose (30 mg/kg) decreased cell proliferation and neurogenesis in the hippocampal DG and SVZ. Our findings show a dissociation between behavioral and proliferative effects of repeated CBD and suggest that the antidepressant-like effects of CBD may occur independently of adult neurogenesis in non-stressed Swiss mice.

Permanent, 3-stage, 4-vessel occlusion as a model of chronic and progressive brain hypoperfusion in rats : a neurohistological and behavioral analysis

Permanent, 3-stage, 4-vessel occlusion (4-VO) was evaluated as a practicable model of progressive, cerebral hypoperfusion in rats, resulting in quantifiable, reproducible, neuronal damage within a time interval shorter than that described in the 2-VO model. The effect of permanent and graded 4-VO on cognition was also evaluated using the newly developed, aversive radial maze. The vertebral arteries (VA) plus the common carotid arteries (CCA) or internal carotid arteries (ICA) were progressively and permanently occluded, following different experimental sequences (CCA--> VA; VA-->CCA-->CCA or VA-->ICA-->ICA) with inter-stage intervals ranging from 1 to 4 weeks. Only two of four groups subjected to 2-stage 4-VO (CCA-->VA) showed modest reduction in the number of normal-appearing CA1 pyramidal cells, despite the significant treatment effect (p < 0.001-0.01 versus sham). A high rate of mortality (63.8%) was associated with 2-stage 4-VO. More pronounced and consistent neuronal damage occurred 8 weeks after 3-stage 4-VO, following the sequence VA --> CCA --> CCA (p < 0.001). One month after this schedule, profound, persistent cognitive impairment was demonstrated in the aversive radial maze (p < 0.01-0.0001). This behavioral effect was not manifested when the ICA, rather than the CCA, were occluded, despite the presence of significant, although less severe, hippocampal lesioning. The mortality rate was significantly reduced when 3-stage 4-VO was used (p < 0.0001). These consistent, histological and behavioral effects, combined with a low mortality rate, suggest that permanent, 3-stage 4-VO may represent a reliable animal model of chronic, progressive, cerebral hypoperfusion.

Cognitive impairment and persistent anxiety-related responses following bilateral common carotid artery occlusion in mice

The present study examined the behavioral and neurohistological changes induced by the bilateral common carotid artery occlusion (BCCAO) model of brain ischemia in Swiss mice. The post-ischemic behavioral effects of 17min BCCAO were recorded 7, 14, and 28 days after reperfusion in the Morris water maze, open field, and elevated plus maze to assess spatial learning and memory, general locomotor activity, and levels of anxiety-like behavior, respectively. After behavioral testing, the brains were removed and processed to evaluate hippocampal neurodegeneration using Nissl staining and Fluoro-Jade C histochemistry and hippocampal neurogenesis using doublecortin immunohistochemistry. BCCAO induced memory impairment 7 and 14 days after reperfusion, with apparent functional recovery 28 days later. Anxiety-related behaviors remained elevated in ischemic compared to sham mice tested 28 days after reperfusion. Hippocampal neurodegeneration was detected in all hippocampal subfields (CA1-CA4) from day 7 to day 28. Decreased hippocampal neurogenesis was observed 14 and 28 days after BCCAO. The effects of BCCAO on spatial memory were transient, whereas anxiety-like behavior was persistent and might be related to CA3 hippocampal injury induced by BCCAO in mice.

Tacrolimus (FK506) reduces ischemia-induced hippocampal damage in rats: a 7- and 30-day study

The neuroprotective effect of the immunosuppressant agent FK506 was evaluated in rats after brain ischemia induced for 15 min in the 4-vessel occlusion model. In the first experimental series, single doses of 1.0, 3.0 or 6.0 mg FK506/kg were given intravenously (iv) immediately after ischemia. In the second series, FK506 (1.0 mg/kg) was given iv at the beginning of reperfusion, followed by doses applied intraperitoneally (ip) 6, 24, 48, and 72 h post-ischemia. The same protocol was used in the third series except that all 5 doses were given iv. Damage to the hippocampal field CA1 was assessed 7 or 30 days post-ischemia on three different stereotaxic planes along the septotemporal axis of the hippocampus. Ischemia caused marked neurodegeneration on all planes (P<0.001). FK506 failed to provide neuroprotection to CA1 both when applied iv as a single dose of 1.0, 3.0 or 6.0 mg/kg (experiment 1), and after five iv injections of 1.0 mg/kg (experiment 3). In contrast, the repeated administration of FK506 combining iv plus ip administration reduced CA1 cell death on all stereotaxic planes both 7 and 30 days post-ischemia (experiment 2; P<=0.01). Compared to vehicle alone, FK506 reduced rectal temperature in a dose-dependent manner (P<=0.05); however, this effect did not alter normothermia (37 C). FK506 reduced ischemic brain damage, an effect sustained over time and apparently dependent on repeated doses and on delivery route. The present data extend previous findings on the rat 4-vessel occlusion model, further supporting the possible use of FK506 in the treatment of ischemic brain damage.

Neurohistological and behavioral changes following the four-vessel occlusion/internal carotid artery model of chronic cerebral hypoperfusion: comparison between normotensive and spontaneously hypertensive rats.

Chronic cerebral hypoperfusion (CCH) may be a prodromal feature of aging-related dementias, and chronic hypertension is a major risk factor. We used a permanent, four-vessel occlusion/internal carotid artery (4-VO/ICA) model to evaluate the cognitive and neurohistological outcomes of CCH in both young and middle-aged rats. Young rats are asymptomatic after permanent 4-VO/ICA, and we tested the hypothesis that chronic hypertension aggravates the outcomes of CCH. Young normotensive rats (NTRs) and young spontaneously hypertensive rats (SHRs) were first subjected to 4-VO/ICA and then examined for hippocampal and cortical neurodegeneration 7, 15, and 30 days later. In a second experiment, both NTRs and SHRs were then trained in a modified, non-food-rewarded aversive radial maze (AvRM) task until acquiring asymptotic performance and then subjected to 4-VO/ICA. Thirty days later, they were assessed for memory retention of the previously acquired cognition. In a third, post hoc experiment, middle-aged NTRs were trained in the AvRM, subjected to 4-VO/ICA, and tested for memory retention 30 days later. Compared with NTRs, both SHRs and middle-aged NRTs had severe hippocampal and cortical damage, but they did not differ from each other, regardless of the chronicity of 4-VO/ICA. In contrast, NTRs were behaviorally asymptomatic, and retrograde memory performance was persistently impaired in SHRs. This amnesic effect in the SHR group was very similar to the middle-aged NTR group. These findings suggest that chronic hypertension deteriorates the capacity of the brain to adaptively respond to CCH. This influence of hypertension may parallel the effect of aging.

Subchronic administration of Trichilia catigua ethyl-acetate fraction promotes antidepressant-like effects and increases hippocampal cell proliferation in mice

ETHNOPHARMACOLOGICAL RELEVANCE Trichilia catigua preparations have been popularly used in Brazil as a tonic for the treatment of fatigue, stress, impotence, and memory deficits. We recently demonstrated an antidepressant-like effect of acute administration of the Trichilia catigua ethyl-acetate fraction (EAF) in mice. The aim of the present study was to evaluate whether subchronic Trichilia catigua EAF administration maintains its antidepressant-like effects and whether these effects are related to hippocampal neurogenesis. MATERIAL AND METHODS Trichilia catigua EAF (200 and 400mg/kg) was orally administered to mice for 14 day. The animals were tested in the forced swim test (FST) or tail suspension test (TST). After behavioral testing, the animals received bromodeoxyuridine (BrdU; 200mg/kg, i.p.) and were euthanized 24h, 7 day, or 15 day later. The brains were assayed for BrdU and doublecortin (DCX) immunohistochemistry to detect cell proliferation/survival and neurogenesis, respectively. RESULTS Subchronic administration of 400mg/kg Trichilia catigua EAF promoted antidepressant-like effects in mice in both the FST and TST. The antidepressant-like effect was accompanied by an increase in cell proliferation in the dentate gyrus (DG) of the hippocampus 24h after the treatments were discontinued. This proliferative effect, however, did not influence cell survival or neurogenesis because no change in the number of BrdU- or DCX-positive cells was detected 7 or 15 day after the last EAF administration compared with controls. CONCLUSIONS Trichilia catigua EAF produced antidepressant-like effects and induced hippocampal cell proliferation in mice. The results contribute information on the pharmacological and molecular mechanisms involved in the antidepressant-like effect of Trichilia catigua EAF.

Ethanol withdrawal activates nitric oxide-producing neurons in anxiety-related brain areas.

The present study investigated whether nitric oxide (NO)-producing neurons localized in brain areas related to anxiety are also activated after ethanol withdrawal. Male Wistar rats were subjected to an oral ethanol self-administration procedure, in which they were offered 6-8% (vol/vol) ethanol solution for a period of 21 days followed by abrupt discontinuation of the treatment. Control animals received control dietary fluid for similar periods of time. Twenty-four or 48 h after ethanol discontinuation, the animals were exposed to the open field for 10 min. Two hours later, their brains were removed and processed for Fos immunohistochemistry and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry (which is used to detect NO-producing neurons). Decreased exploratory activity was observed in animals subjected to 24-h withdrawal, characterized by a shorter distance traveled in the open field. Additionally, increased Fos expression was detected in brain areas, such as the cingulate and piriform cortices, several hypothalamic nuclei, amygdaloid nuclei, most subdivisions of the periaqueductal gray matter, and dorsal raphe nucleus (DRN). Ethanol withdrawal activated NO-producing neurons in the paraventricular nucleus (PVN) of the hypothalamus, dorsolateral periaqueductal gray matter (DLPAG), and DRN. The results show that ethanol withdrawal activates NO-producing neurons in the PVN, DLPAG, and DRN, which are brain areas implicated in the modulation of emotional, autonomic, and motor expression of anxiety-like behaviors.

The cognitive and histopathological effects of chronic 4-vessel occlusion in rats depend on the set of vessels occluded and the age of the animals.

Continuing previous efforts to develop the 4-vessel occlusion (4-VO) model of chronic cerebral hypoperfusion (CCH), here we evaluated whether permanent, stepwise 4-VO causes both learning deficits, hippocampal neurodegeneration and retinal lesion in young, middle-aged or aged rats. Chronic 4-VO was induced by ligation of different sets of vessels, i.e., the vertebral arteries (VA) plus common carotid arteries (CCA) (4-VO/CCA model) or the VA plus internal carotid arteries (ICA) (4-VO/ICA model) with a 1-week interstage interval. Forty days after the 4-VO, the rats were tested for spatial learning impairment, and then examined for hypoxic/ischemic damage. Young, 4-VO/CCA rats exhibited cognitive impairment, hippocampal neurodegeneration and retinal lesion (p<0.0001-0.05). After 4-VO/ICA, neither young nor middle-aged rats exhibited any learning deficits, hippocampal or retinal damage. In aged rats, chronic 4-VO/ICA caused a mild learning deficit (p<0.05). A significant effect of training was observed for the old, sham-operated rats (p<0.0001-0.001), but not for the aged 4-VO/ICA rats (p>0.05). On average, hippocampal cell density did not change after 4-VO/ICA in aged rats, but 3 of 10 subjects exhibited reduced pyramidal cell counts in all hippocampal subfields. Retinal morphology appeared to be unaffected in the 4-VO/ICA aged rats. These data suggest that the 4-VO/ICA model, but not the 4-VO/CCA model, is a suitable paradigm to study the behavioral outcome of CCH given the preservation of the retina after 4-VO/ICA. Moreover, the age at which 4-VO/ICA occurs seems to be an important factor for determining the behavioral and neuropathological changes.