Teresa Fortin

Chronic reduction of cerebral blood flow in the adult rat: late-emerging CA1 cell loss and memory dysfunction

Ten-month-old rats were subjected to permanent bilateral occlusion of both common carotid arteries (2-VO) to chronically but moderately reduce brain blood flow. 2-VO impaired Morris water maze acquisition as soon as 7 days post-surgery. 2-VO also caused a later-appearing impairment on the radial arm maze which did not reach significance until 63 days post-surgery. At 14 dats post-surgery there were no effects of 2-VO on hippocampal CA1 pyramidal cell number or density of glial fibrillary acidic protein (GFAP). Hippocampal choline acetyltransferase activity at 70 days was also unaffected by 2-VO. At 190 days post-surgery, however, the 2-VO rats showed loss of cells and increased GFAP density in CA1. The increased density of hippocampal GFAP correlated with radial arm maze but not Morris water maze impairment. It is suggested that 2-VO causes neuronal dysfunction which can be exacerbated by stress and thereby manifested on aversively motivated tasks such as the water maze. As well, CA1 neurons begin to degenerate after several weeks of the reduced energy availability caused by 2-VO and this impairs memory. Since reduced neuronal energy metabolism is associated with the progressive neurodegeneration that underlies disorders such as Alzheimers, research should further explore the possibility that the effects of 2-VO may model age-related dementia.

Cleavage of amyloid precursor protein elicited by chronic cerebral hypoperfusion

In the present study, we sought to determine whether low-grade, chronic vascular insufficiency induced in a rodent model of chronic cerebrohypoperfusion is sufficient, in and of itself, to trigger cleavage of the amyloid precursor protein (APP) into beta A-sized fragments. We report that chronic two vessel occlusion (2VO) results in progressive accumulation of beta A peptides detected by Western analysis in aged rats correlating with a shift in the immunohistochemical localization of APP from neurons to extracellular deposits in brain parenchyma. These data indicate that the 2VO paradigm reproduces features of beta A biogenesis characteristic of sporadic Alzheimers disease.

Chronic cerebral hypoperfusion elicits neuronal apoptosis and behavioral impairment

CHRONIC reductions in cerebral blood flow associated with aging and progressive neurodegenerative disorders can precipitate cognitive failure. To assess whether chronic cerebrovascular insufficiency elicits neuronal apoptosis, apoptotic cell death in the hippocampus was quantitated in a rat model of permanent carotid occlusion. Bilateral carotid artery occlusion (2VO) was shown to induce apoptotic morphology and DNA strand breaks in hippocampal neurons 2 and 27 weeks after ligation. The rate of pyramidal cell apoptosis was higher at chronic (27 weeks) compared to sub-chronic (2 weeks) time points. 2VO-induced apoptosis resulted in a decrease in total pyramidal cell number at 27 weeks but not at earlier time points, indicating progressive neuronal loss. Working and reference memory errors in the radial arm maze were strongly correlated with the number of apoptotic neurons in CA1 but not CA3 pyramidal cell fields. These data provide the first indication that apoptotic loss of pyramidal neurons may play a role in memory impairment associated with clinical conditions of chronic cerebrovascular insufficiency.

Perinatal manganese exposure: Behavioral, neurochemical, and histopathological effects in the rat

Manganese chloride (Mn) was dissolved in the drinking water (0, 2, or 10 mg/ml) of dams and their litters from conception until postnatal day (PND) 30. Parturition was uneventful in the Mn-exposed rats and no physical abnormalities were observed. The rats exposed to 10 mg/ml Mn showed a 2.5-fold increase in cortical Mn levels. Their weight gain was attenuated from PND 9-24 and they were hyperactive at PND 17. Neither the 2 nor the 10 mg/ml Mn-exposed groups differed from the controls on the elevated plus apparatus or on the Morris water maze and the radial arm maze. Brain monoamine levels and choline acetyltransferase activity were affected. Tyrosine hydroxylase immunohistochemistry showed that dopamine cells of the substantia nigra were intact. Glial fibrillary acidic protein immunoreactivity was not increased in cortex, caudate, and hippocampus. However, both the low- and high-dose Mn-exposed groups showing thinning of the cerebral cortex. This could have resulted from perinatal malnutrition or from a direct effect of Mn on cortical development.

Hippocampal nitric oxide upregulation precedes memory loss and Aβ 1-40 accumulation after chronic brain hypoperfusion in rats

Abstract Chronic brain hypoperfusion (CBH) using permanent occlusion of both common carotid arteries in an aging rat model, has been shown to mimic human mild cognitive impairment (MCI), an acknowledged high risk condition that often converts to Alzheimers disease. An aging rat model was used to determine whether hippocampal nitric oxide (NO) is abnormally expressed following CBH for two or eight weeks. At each time point, spatial memory was measured with the Morris water maze and hippocampal Aβ 1-40/1-42 concentrations were obtained using sandwich ELISA. Real-time amperometric measures of NO representing the constitutive isoforms of neuronal nitric oxide synthase (nNOS) and endothelial (e)NOS were also taken at each time point to ascertain whether NO levels changed as a result of CBH, and if so, whether such NO changes preceded or followed any memory or amyloid-beta pathology. We found that two weeks after CBH, NO hippocampal levels were upregulated nearly four-fold when compared to nonoccluded rats but no alteration in spatial memory of Aβ products were observed at this time point. By contrast, NO concentration had declined to control levels by eight weeks but spatial memory was found significantly impaired and Aβ 1-40 (but not Aβ 1-42) had increased in the CBH group when compared to control rats. Since changes in shear stress are known to upregulate eNOS but generally not nNOS, these results suggest that shear stress induced by CBH hyperactivated vascular NO derived from eNOS in the first two weeks as a reaction by the capillary endothelium to maintain homeostasis of local cerebral blood flow. The return of vascular NO to basal levels after eight weeks of CBH may have triggered metabolic changes within hippocampal cells resulting in hippocampal dysfunction as reflected by spatial memory impairment and by accumulation of Aβ 1-40 peptide. In conclusion, our study shows that CBH initiates spatial memory loss in aging rats thus mimicking human MCI and also increases Aβ 1-40 in the hippocampus. The memory and amyloid changes are preceded by NO upregulation in the hippocampus. These preliminary findings may be important in understanding, at least in part, the molecular mechanisms that precede memory impairment during chronic brain ischemia and as such, the pre-clinical stage leading to Alzheimers disease.

Retinal and Optic Nerve Degeneration After Chronic Carotid Ligation Time Course and Role of Light Exposure

Background and Purpose— Carotid artery disease can cause chronic retinal ischemia, resulting in transient or permanent blindness, pupillary reflex dysfunction, and retinal degeneration. This experiment investigated the effects of chronic retinal ischemia in an animal model involving permanent carotid occlusion. The time course of retinal pathology and the role of light in this pathology were examined. Methods— Sprague-Dawley rats underwent permanent bilateral occlusion of the common carotid arteries or sham surgery. Half of the animals were postsurgically housed in darkness, and half were housed in a 12-hour light/dark cycle. Animals were killed at 3, 15, and 90 days after surgery. Stereological techniques were used to count the cells of the retinal ganglion cell layer. Thy-1 immunoreactivity was assessed to specifically quantify loss of retinal ganglion cells. The thicknesses of the remaining retinal sublayers were measured. Optic nerve degeneration was quantified with the Gallyas silver staining technique. Results— Permanent bilateral occlusion of the common carotid arteries resulted in loss of the pupillary reflex to light in 58% of rats. Eyes that lost the reflex showed (1) optic nerve degeneration at 3, 15, and 90 days after surgery; (2) a reduction of retinal ganglion cell layer neurons and Thy-1 immunoreactivity by 15 and 90 days; and (3) a severe loss of photoreceptors by 90 days when postsurgically housed in the light condition only. Conclusions— Ischemic damage to the optic nerve caused loss of pupillary reflex and death of retinal ganglion cells in a subset of rats. Subsequently, light toxicity induced death of the photoreceptors.

Chronic cerebral hypoperfusion: loss of pupillary reflex, visual impairment and retinal neurodegeneration.

Adult rats underwent permanent bilateral occlusion of the common carotid arteries (2VO) to determine the effect of chronic cerebral ischemia on vision and retina. They were monitored post-surgically for the presence of the pupillary reflex to light. Some rats were tested for 6 months post-surgically on a radial arm maze task and then tested in another water-escape task which explicitly tested visual function. Another group of rats were tested post-surgically for 3 months on a task which simultaneously assessed visual and tactile discrimination ability. The thicknesses of the retinal sub-layers were then measured for some rats. Fourteen of the 25 rats that underwent 2VO lost the pupillary reflex. This seemed to occur within 5 days. Rats that lost the pupillary reflex but not rats whose reflex was intact, were impaired on all visually guided mazes. Tactile discrimination ability was unaffected. Only rats that lost the pupillary reflex showed reduced thickness of the retinal outer nuclear and plexiform layers, reduced cell density in the retinal ganglion cell layer and astrocytosis and degeneration of the optic tract. We conclude that 2VO can eliminate the pupillary reflex. Photoreceptors and retinal ganglion cells degenerate, but it is unclear if these are the cause(s) or result(s) of the loss of the pupillary reflex. These effects are accompanied by impairment of visually guided behavior. The possibility that visual system damage may also occur in acute ischemia merits further investigation.

192 IgG-saporin lesion of basal forebrain cholinergic neurons in neonatal rats

Seven day old rats received bilateral intraventricular injections (200 ng) of the immunotoxin 192 IgG-saporin. When assayed in adulthood, these rats showed an 84% loss of hippocampal and a 52% loss of cortical choline acetyltransferase (ChAT) activity. ChAT was unaffected in the caudate. Cholinergic neurons immunoreactive (IR) for the low affinity neurotrophin receptor (P75NTR) were severely reduced throughout the basal forebrain nuclei. Cortical and hippocampal norepinephrine were increased and these areas showed ingrowth of ectopic, P75NTR and dopamine beta-hydroxylase IR varicosities. These were probably sympathetic axons. No obvious forebrain dysmorphogenesis was observed and cortical thickness was unaffected. These rats showed no evidence of impaired spatial learning/memory as assessed by the Morris water maze and delayed spatial alternation. However, they were less active on the elevated plus apparatus and spent less time on the open arms, suggestive of increased timidity. 192 IgG-saporin appears to be a powerful tool to selectively lesion basal forebrain cholinergic neurons in the neonatal rat. Surprisingly, the neuromorphological and behavioral sequelae seem minimal. It may be necessary to achieve near-total neonatal destruction of forebrain cholinergic neurons before severe, lasting mnemonic effects are evident.

A chronic physiological rat model of dementia

We have developed an aging rat model that mimics specific pathology reported in dementia, particularly Alzheimers disease (AD). The model involves subjecting rats to chronic cerebrovascular insufficiency (CVI) for 1-9 weeks. Gross and sensory-motor function remains normal but spatial memory acquisition and retention are impaired after 1 week and worsens progressively with time. In vivo [31P]NMR spectroscopy evaluation in CVI animals showed membrane phospholipid synthesis increase in the hippocampal-cortex region of affected animals which increases with time. Post-mortem examination revealed that CA1 neurons can express selective damage 1 week after CVI and the number of CA1 neurons thus affected increases in proportion with time. MOreover, there is progressive increase in GFAP hypertrophy and hyperplasia in the hippocampal region during the 9-week observation period. Reduction of microtubule-associated protein 2 and pre-terminal noradrenergic varicosities in the hippocampus-cortex is seen after 9 weeks but not 1 week of CVI. All the above changes have been reported in AD-affected brains. The present CVI model appears as a useful screen in investigating potential therapy for AD as well as increasing understanding of this disorder.

Pinealectomy: behavioral and neuropathological consequences in a chronic cerebral hypoperfusion model

This experiment determined if pinealectomy (PX) affects the consequences of chronic, moderate brain ischemia. Rats were pinealectomized at 25 days of age and trained at 9 months on a tactile radial maze. They then underwent permanent occlusion of the common carotid arteries (2VO) or sham surgery, followed by maze retraining and then neurohistological assessment at 16 months. Combined PX + 2VO rats committed more working memory errors on the maze. 2VO itself caused a 10% reduction in hippocampal CA1 pyramidal cell number. PX alone caused a 21% reduction. Combined PX and 2VO caused the greatest reduction (32%) of CA1 cells. Similar results were seen for CA4. PX also increased glial fibrillary acidic protein immunoreactivity in both CA1 and CA4. Thus PX not only augmented the consequences of chronic brain ischemia but notably, PX itself caused hippocampal damage. These effects seemed not to result from the small cortical lesion caused by the PX procedure. The results are consistent with the hypothesis that endogenous melatonin is a neuroprotectant in the aging brain.