Bihemispheric reduction of GABAA receptor binding following focal cortical photothrombotic lesions in the rat brain.

Focal brain lesions may lead to neuronal dysfunctions in remote (exofocal) brain regions. In the present study, focal lesions were induced in the hindlimb representation area of the parietal cerebral cortex in rats using the technique of photothrombosis. Photothrombosis occurs after illumination of the brain through the intact skull following intravenous injection of the photosensitive dye Rose Bengal. This resulted in cortical lesions with a diameter of about 2 mm. Quantitative receptor autoradiography was used to study alterations in the density of [3H]muscimol binding sites to GABAA receptors seven days after lesion induction. A reduced GABAA receptor binding (-13 to -27% of the control value) was found in layers II and III of remote exofocal regions in the ipsi- and contralateral cortex. The reduction was consistently more intense in the ipsilateral cortical areas than in those of the contralateral hemisphere. Using extracellular recordings, significant correlations between GABAA receptor binding and paired pulse inhibition could be demonstrated. The present investigation demonstrates that focal brain lesions cause a widespread, functionally effective down-regulation of GABAA receptors. These postlesional changes may result from lesion-induced alterations in cortical connectivity.

Epileptiform discharges to extracellular stimuli in rat neocortical slices after photothrombotic infarction.

Seizures are one of the most frequent complications after cerebral ischemia in patients. Up to now it is unknown which mechanisms are responsible for this. As shown previously photothrombotic infarction in rat neocortex leads to a sweeping suppression of GABAergic inhibition. In this study we investigated whether and to what extent epileptiform discharges can be observed in this ischemia model. In neocortical slices from lesioned animals we did not find spontaneous epileptic activity or paroxysmal depolarisation shifts. However, ipsi- and contralateral to a photothrombotic lesion the frequency of double and multiple discharges was markedly increased when compared to unlesioned controls. Surprisingly, neither the drug lubeluzole which was has been shown to prevent the GABAergic disinhibition observed after photothrombotic lesioning of rat neocortex, nor the prevention of spreading depressions by the NMDA-receptor antagonist MK-801 during lesion induction significantly affected the frequency of epileptiform discharges. This indicates that the epileptiform discharges are probably caused by functional alterations of glutamatergic receptors.

Transient changes in the presence of nitric oxide synthases and nitrotyrosine immunoreactivity after focal cortical lesions.

Since ischemic insults lead to a deregulation of nitric oxide production which contributes to delayed neuronal death, we investigated changes in the distribution and amount of nitric oxide synthases I and II and in the appearance of nitrotyrosine caused by small, well-defined photothrombic lesions (2 mm in diameter) in the somatosensory cortex of rats. Four hours after lesioning, cell loss was evident in the core of the lesion and no nitric oxide synthase was present within this area, indicating that neurons expressing nitric oxide synthase I were lost or that nitric oxide synthase I was degraded. No increase in the number of neurons expressing nitric oxide synthase I was visible in the area surrounding the lesion, nor in other parts of the brain. One day after lesioning, NADPH-diaphorase- and nitric oxide synthase II-positive leucocytes had invaded the perilesional cortex and were accumulated in injured blood vessels. By two to three days post-lesion, layer V and VI pyramidal neurons, microglia, astrocytes and invading leucocytes had become strongly immunoreactive for nitric oxide synthase II within a perilesional rim. The number of cells expressing nitric oxide synthase I remained stable. Nitric oxide synthase II immunoreactivity and related NADPH-diaphorase had decreased by seven days post-lesion in most animals. However, the number of activated microglia or macrophages and astrocytes, as revealed by other markers, remained elevated. In addition, nitrotyrosine immunoreactivity was evident in the blood vessels close to the lesion, as well as in the ipsilateral hippocampus and thalamus. These findings indicate that no perilesional changes in the number of neurons expressing nitric oxide synthase I occur, but that a transient increase in nitric oxide synthase II does take place in the aftermath of small cortical lesions. The results suggest that increased nitric oxide production is limited to certain post-lesional intervals in this experimental model. It is also obvious that the vast majority of nitric oxide synthase-positive cells are nitric oxide synthase II-containing astrocytes three days after lesioning, suggesting that astrocyte-derived nitric oxide plays a significant role in delayed neuronal death. Such a condition points to an important aspect of post-lesional astrocytosis.

Copper-zinc superoxide dismutase and isolectin B4 binding are markers for associative and transhemispheric diaschisis induced by focal ischemia in rat cortex.

Copper/zinc-superoxide dismutase (Cu/Zn-SOD) belongs to a class of enzymes, identified as essential and highly effective endogenous scavengers of cytotoxic oxygen radicals. These radicals contribute to postlesional neurotoxicity. In order to determine the superoxide-scavenging potential of regions affected by unilateral cortical photothrombosis, we studied the changes in the distribution of Cu/Zn-SOD and the appearance of activated microglia by immunohistochemistry and isolectin B4 binding. Four hours postlesion, Cu/Zn-SOD increased significantly within a homotopic area of the contralateral hemisphere and in ipsilateral thalamic nuclei, whereas isolectin B4-positive microglia were upregulated at days 5 and 7 postlesion within the same regions. The contralateral increase in the amount of the superoxide-scavenging Cu/Zn-SOD indicates that this enzyme is induced by a retrograde reaction carried through callosal connections.

Pharmacological reduction of electrophysiological diaschisis after photothrombotic ischemia in rat neocortex.

Focal cerebral lesions in the rat brain induced by photothrombosis cause hyperexcitability of the surrounding brain. This can be demonstrated in brain slices taken from animals several days after lesioning, by analysis of field potential responses to paired-pulse stimulation. We now investigated whether and how these remote effects of a cortical lesion can be modified pharmacologically. Application of the NMDA receptor antagonist, MK-801 ((+)-5-methyl-10, 11-dihydro-5H-dibnzo[a,d]cyclohepten-5,10-imine), was shown to block induction of immediate early genes and activation of astrocytes as evidenced by glial fibrillary acidic protein (GFAP) staining in the photothrombosis model. However, MK-801 did not affect the hyperexcitability that had been demonstrated by field potential recordings in brain slices. In another series of experiments, lubeluzole ((+)-(S)-4-(2-benzothiazolylmethylamino)-alpha-[(3,4-difluoroph enoxy) methyl]-1-piperidineethanol), which inhibits the glutamate-activated nitric oxide pathway as evidenced by down-regulation of intracellular cyclic GMP, was given immediately after induction of the insult. This reduced hyperexcitability as investigated 7 days later. In the light of these data one can suggest that a nitric oxide-cyclic GMP-related mechanism may be responsible for functional alterations in the surround of photothrombotic brain lesions.

Extended brain disinhibition following small photothrombotic lesions in rat frontal cortex.

The effect of an ischaemic focal cortical lesion on the excitability of surrounding and remote brain areas was investigated. Infarcts were produced photothrombotically in rat frontal cortex and brain excitability was assessed by a extracellular paired-pulse stimulation in coronal slices 7 days later. The cortical lesions caused a reduction of inhibition. The extent and grade of these electrophysiological effects depended on the depth of the lesion: in animals with a lesion affecting the deeper cortical layers a pronounced transcortical diaschisis was found, whereas animals with a shallow lesion showed only a slight ipsilateral affliction. The study shows that focal lesions in the motor cortex cause widespread disinhibition, probably resulting from deafferentation, and these may have a significant impact on recovery of function.

Systemically administered cycloheximide reduces inhibition in rat neocortical slice preparation.

Global cerebral ischemia leads to long lasting hyperexcitability and a reduced protein synthesis in non-infarcted tissue surrounding the lesion. In this study we investigated whether protein synthesis inhibition by pharmacological means itself changes neocortical excitability. Two hours after the last of three i.p. injections with the protein synthesis inhibitor cycloheximide (12 h interval, 1.5 mg/kg body weight) we observed a widespread reduction of neocortical inhibition. The study indicates that inhibition of protein synthesis may contribute to the altered brain excitability following ischemia.

Electrophysiological transcortical diaschisis after cortical photothrombosis in rat brain.

BACKGROUND AND PURPOSE: The severity of functional deficits after a cortical infarction often does not correlate with lesion size. The stroke may affect pathways connecting to distant brain regions and therefore may also alter the function of remote parts of the cortex. Remote changes in electric activity, blood flow, and metabolism are called diaschisis. In the present study we addressed the question of whether in brain areas contralateral to a photochemically induced cortical infarction alteration of excitability can be observed as an indication of the effects of diaschisis. METHODS: We induced focal lesions in the sensory area at the border of the motor and occipital cortices by injecting the photosensitizing dye rose bengal and illuminating the skull stereotaxically. Seven days after induction of photothrombosis, electrophysiological recordings were obtained with standard methods from 400-microns-thick neocortical coronal slices. As an indication of inhibition we used a paired-pulse stimulus protocol and calculated a ratio of the amplitudes of the second versus the first excitatory postsynaptic potential. RESULTS: In lesioned animals we found a significant increase of the ratio over a wide zone of the neocortex, both ipsilateral and contralateral, compared with unlesioned animals. CONCLUSIONS: Our results suggest that a neocortical infarction leads to hyperexcitability not only in its direct vicinity but also in the contralateral hemisphere. Such hyperexcitability may contribute to increased activation of contralateral brain areas and to functional reorganization after stroke.