András Mihály

Experimental cerebral hypoperfusion induces white matter injury and microglial activation in the rat brain

Though cerebral white matter injury is a frequently described phenomenon in aging and dementia, the cause of white matter lesions has not been conclusively determined. Since the lesions are often associated with cerebrovascular risk factors, ischemia emerges as a potential condition for the development of white matter injury. In the present study, we induced experimental cerebral hypoperfusion by permanent, bilateral occlusion of the common carotid arteries of rats (n=6). A sham-operated group served as control (n=6). Thirteen weeks after the onset of occlusion, markers for astrocytes, microglia, and myelin were found to be labeled by means of immunocytochemistry in the corpus callosum, the internal capsule, and the optic tract. The ultrastructural integrity and oligodendrocyte density in the optic tract were investigated by electron microscopy. Quantitative analysis revealed that chronic cerebral hypoperfusion caused mild astrogliosis in the corpus callosum and the internal capsule, while astrocytic disintegration in the optic tract increased by 50%. Further, a ten-fold increase in microglial activation and a nearly doubled oligodendrocyte density were measured in the optic tract of the hypoperfused rats as compared with the controls. Finally, vacuolization and irregular myelin sheaths were observed at the ultrastructural level in the optic tract. In summary, the rat optic tract appears to be particularly vulnerable to ischemia, probably because of the rat brain’s angioarchitecture. Since the detected glial changes correspond with those reported in vascular and Alzheimer dementia, this model of cerebral hypoperfusion may serve to characterize the causal relationship between ischemia and white matter damage.

Diazoxide and dimethyl sulphoxide prevent cerebral hypoperfusion-related learning dysfunction and brain damage after carotid artery occlusion

Chronic cerebral hypoperfusion, a mild ischemic condition is associated with advancing age and severity of dementia; however, no unanimous therapy has been established to alleviate related neurological symptoms. We imposed a permanent, bilateral occlusion of the common carotid arteries of rats (n=18) to create cerebral hypoperfusion. A mitochondrial ATP-sensitive K+ channel opener diazoxide (DZ, 5 mg/kg) or its solvent dimethyl sulphoxide (DMSO) were administered i.p. (0.25 ml) on five consecutive days after surgery. Sham-operated animals (n=18) served as control for the surgery, while nontreated rats were used as control for the treatments. Three months after the onset of cerebral hypoperfusion, the rats were tested in a hippocampus-related learning paradigm, the Morris water maze. Subsequently, the animals were sacrificed and neurons, astrocytes and microglia were labeled with immunocytochemistry in the dorsal hippocampus. DMSO and diazoxide dissolved in DMSO restored cerebral hypoperfusion-related learning dysfunction and prevented cyclooxygenase-2-positive neuron loss in the dentate gyrus. Cerebral hypoperfusion led to reduced astrocyte proliferation, which was not clearly affected by the treatment. Microglia activation was considerably enhanced by cerebral hypoperfusion, which was completely prevented by diazoxide dissolved in DMSO, but not by DMSO alone. We conclude that diazoxide can moderate ischemia-related neuroinflammation by suppressing microglial activation. Furthermore, we suggest that DMSO is a neuroprotective chemical in ischemic conditions, and it must be considerately used as a solvent for water-insoluble compounds in experimental animal models.

Age-related microvascular degeneration in the human cerebral periventricular white matter.

Clinical studies have identified white matter (WM) lesions as hyperintensive regions in the MRI images of elderly patients. Since a cerebrovascular origin was attributed to such lesions, the present analysis set out to define the microvascular histopathologic changes in the periventricular WM in the aged. Post-mortem samples of the frontal, parietal, and occipital periventricular WM of 40–90-year-old subjects were prepared for quantitative light and electron microscopy. Light microscopic examination revealed microvascular fibrohyalinosis as the most common type of microvascular damage in the elderly. Ultrastructural analysis identified the microvascular thickening as collagen deposits affecting the basement membrane. The vascular density did not correlate with the age. The basement membrane pathology significantly increased, while the number of intact microvessels gradually decreased, with advancing age in the frontal and occipital WM. Finally, peripheral atherosclerosis coincided with massive microvascular fibrosis, particularly in the frontal WM. Our results demonstrate an age-related microvascular degeneration in the periventricular WM, which may contribute to the development of WM lesions by hindering a sufficient supply of nutrients to the affected WM sites. Furthermore, the data accord with previous observations identifying the frontal lobe as the site at which WM vulnerability is most pronounced. Finally, atherosclerosis in large, peripheral vessels is considered to be a predictive marker of microvascular pathology in the WM.

Immunohistochemical localization of extravasated serum albumin in the hippocampus of human subjects with partial and generalized epilepsies and epileptiform convulsions

SummaryHuman serum albumin-like immunoreactivity was detected by the peroxidase-antiperoxidase method in histological sections of the hippocampus from epileptic and control brains obtained on routine autopsies. In the hippocampi of epileptic patients immunoreactive astrocytes were found, the number of which was increasing with the severity of the manifest convulsions. The highest numbers of immunoreactive astrocytes were observed in those patients who died in status epilepticus. Hippocampi from control patients with no neurologic disorders in life were devoid of immunoreactive astrocytes. The results are discussed in terms of the breakdown of the blood-brain barrier during epileptic seizures.

Topography and associations of luteinizing hormone–releasing hormone and neuropeptide Y–immunoreactive neuronal systems in the human diencephalon

Neuropeptide Y (NPY) potentiates the effect of luteinizing hormone–releasing hormone (LHRH) on luteinizing hormone secretion in several species, including human. In addition to the pituitary sites, the interactions of the NPY and LHRH systems may involve diencephalic loci. However, the morphologic basis of this putative communication has not yet been elucidated in the human brain. To discover interaction sites, the distribution and connections of LHRH and NPY‐immunoreactive (IR) neuronal elements in the human hypothalamus were investigated by means of light microscopic single‐ and double‐label immunocytochemistry. NPY‐IR perikarya and fibers were found to be widely distributed in the ventral diencephalon, with high densities in the preopticoseptal, periventricular, and tuberal regions. Small neuronal cell groups were infiltrated with a dense network of varicose NPY‐IR fibers in the lateral preoptic area. The LHRH‐IR perikarya were located mainly in the preopticoseptal region, diagonal band of Broca, lamina terminalis, and periventricular and infundibular nuclei. A few LHRH‐IR neurons and fibers were scattered in the mamillary region. The overlap between the NPY and LHRH systems was apparent in the periventricular, paraventricular, and infundibular nuclei. Double‐labeling immunohistochemistry showed NPY‐IR axon varicosities in contact with LHRH‐IR perikarya and main dendrites. The putative innervation of LHRH neurons by NPY‐IR fibers was also seen in 1‐μm‐thick plastic sections and with confocal laser scanning microscope, thus further supporting the functional impact of NPY‐IR terminals on LHRH‐IR neurons. The present findings suggest that the hypophysiotropic LHRH‐synthesizing neurons may be innervated by intrahypothalamic NPY‐IR fibers. Confirmation by ultrastructural analysis would demonstrate that the LHRH system in the human hypothalamus is regulated by NPY, as has been demonstrated in nonhuman species. J. Comp. Neurol. 427:593–603, 2000.

The kynurenate analog SZR-72 prevents the nitroglycerol-induced increase of c-fos immunoreactivity in the rat caudal trigeminal nucleus: Comparative studies of the effects of SZR-72 and kynurenic acid

Administration of nitroglycerol in a migraine model results in an increased number of c-fos-expressing secondary sensory neurons in the caudal trigeminal nucleus. Since synapses between first- and second-order trigeminal neurons are mediated by excitatory amino acids, NMDA receptors are inhibited by kynurenic acid, though this crosses the blood-brain barrier only poorly. Systemic treatment of rats with SZR-72, a newly synthetized kynurenic acid analog, diminished the nitroglycerol-induced increase of c-fos immunoreactivity in the brain stem highly significantly, while treatment with kynurenic acid resulted in a significantly smaller decrease, proving that SZR-72 is much more effective than kynurenic acid.

Beta-amyloid peptide-induced blood-brain barrier disruption facilitates T-cell entry into the rat brain.

Activated T-lymphocytes can migrate through the blood-brain barrier (BBB) and are able to invade the central nervous system (CNS). In the present study, we investigated whether disruption of the BBB leads to enhanced T-cell migration into the CNS. Amyloid-beta peptide 25-35 (A beta) or tumor necrosis factor-alpha (TNFalpha) were administered into the right common carotid artery of adult male Wistar rats. The agents were administered either alone, or were followed by a cell suspension of exogenously activated T-cells. Rats of other groups received activated or non-stimulated T-lymphocytes only. Sagittal brain sections were analyzed with immunohistochemistry of CD3 to reveal the presence of T-lymphocytes within the CNS parenchyma. Administration of activated T-cells alone led to T-cell migration into the brain. Infusion of either substances (A beta or TNFalpha) resulted in T-cell invasion of the CNS even when no exogenous T-cells were added. Infusion of either of the agents together with T-lymphocytes generated a more intense T-lymphocyte migration than in the other groups. Electron microscopic analysis and Evans-blue extravasation studies confirmed parallel disruption of the BBB. Our study demonstrates that A beta and TNFalpha induce enhanced T-lymphocyte migration towards the brain. This effect may be attributed at least partly to dysfunctioning of the BBB, but other mechanisms are also possible.

Kynurenine in combination with probenecid mitigates the stimulation-induced increase of c-fos immunoreactivity of the rat caudal trigeminal nucleus in an experimental migraine model

SummaryNitroglycerin, often used as a migraine model, results in increased number of c-fos immunoreactive secondary sensory neurons in the caudal trigeminal nucleus. Since synapses between first- and second-order trigeminal neurons are mediated by excitatory amino acids, NMDA receptors are presumably inhibited by kynurenic acid, the only known endogeneous NMDA receptor antagonist. Although kynurenic acid does not cross the BBB, its precursor, kynurenine, if combined with probenecid, crosses it readily. Systemic kynurenine + probenecid treatment significantly diminishes nitroglycerin-induced increase of c-fos immunoreactivity in the brainstem.

The effect of pre- and posttreatment with diazoxide on the early phase of chronic cerebral hypoperfusion in the rat

Diazoxide has been identified as a mitochondrial, ATP-dependent K(+) channel opener, and a potentially neuroprotective compound under ischemic conditions. We set out to characterize the consequences of various treatment strategies with diazoxide in a rat model of chronic cerebral hypoperfusion. Cerebral hypoperfusion was induced by permanent, bilateral occlusion of the common carotid arteries (2VO, n = 36), sham-operated rats serving as controls (SHAM, n = 29). Diazoxide or its vehicle was administered i.p. daily (5 x 0.5 mg/kg/0.25 ml) or as a bolus injection (5 mg/kg/0.25 ml) before surgery or daily after surgery (5 x 0.5 mg/kg/0.25 ml). Spatial learning performance was assessed 1 week after 2VO in the Morris maze. Hippocampal pyramidal cell loss was assessed on cresyl violet-stained sections, while glial reactivity was labeled immunocytochemically. Daily or bolus pretreatment with diazoxide significantly improved 2VO-related learning impairment, whereas posttreatment was ineffective. The number of CA1 pyramidal neurons was reduced by 2VO, which was prevented by repeated or bolus pretreatment with diazoxide. Astrocyte proliferation and microglial activation were enhanced by posttreatment with diazoxide in the hippocampus CA1 area of 2VO animals as compared with SHAM. These data demonstrate that the neuroprotective effect exerted by diazoxide depends on the time of administration with respect to the onset of ischemia; pretreatment but not posttreatment with the compound has proved to be neuroprotective in chronic cerebral hypoperfusion. Thus, pretreatment with diazoxide offers therapeutical prospects for the treatment of cerebral ischemia.

Naltrexone potentiates 4-aminopyridine seizures in the rat

The effects of a pharmacological blockade of the mu opiate receptors on the manifestation of tonic-clonic seizures were investigated in freely moving animals. 4-aminopyridine, a specific blocker of the neuronal K+ channels was used to produce generalized convulsions. After pretreatment of adult rats with 1 mg/kg naltrexone HCl, 3, 5, 7, 9, 14 mg/kg 4-aminopyridine was injected intraperitoneally, and the latencies of the symptoms generated by 4-aminopyridine were measured. Naltrexone HCl decreased these latencies and enhanced the seizures significantly. The experiments provided further evidence for the existence of a tonic anticonvulsant opioid system in the brain.