Intracerebral Injections Of Kainic Acid Research Articles

Caspase 3 involves in neuroplasticity, microglial activation and neurogenesis in the mice hippocampus after intracerebral injection of kainic acid

BackgroundThe roles of caspase 3 on the kainic acid-mediated neurodegeneration, dendritic plasticity alteration, neurogenesis, microglial activation and gliosis are not fully understood. Here, we investigate hippocampal changes using a mouse model that receive a single kainic acid-intracerebral ventricle injection. The effects of caspase 3 inhibition on these changes were detected during a period of 1 to 7 days post kainic acid injection.ResultNeurodegeneration was assessed by Fluoro-Jade B staining and neuronal nuclei protein (NeuN) immunostaining. Neurogenesis, gliosis, neuritic plasticity alteration and caspase 3 activation were examined using immunohistochemistry. Dendritic plasticity, cleavvage-dependent activation of calcineurin A and glial fibrillary acidic protein cleavage were analyzed by immunoblotting. We found that kainic acid not only induced neurodegeneration but also arouse several caspase 3-mediated molecular and cellular changes including dendritic plasticity, neurogenesis, and gliosis. The acute caspase 3 activation occurred in pyramidal neurons as well as in hilar interneurons. The delayed caspase 3 activation occurred in astrocytes. The co-injection of caspase 3 inhibitor did not rescue kainic acid-mediated neurodegeneration but seriously and reversibly disturb the structural integrity of axon and dendrite. The kainic acid-induced events include microglia activation, the proliferation of radial glial cells, neurogenesis, and calcineurin A cleavage were significantly inhibited by the co-injection of caspase 3 inhibitor, suggesting the direct involvement of caspase 3 in these events. Alternatively, the kainic acid-mediated astrogliosis is not caspase 3-dependent, although caspase 3 cleavage of glial fibrillary acidic protein occurred.ConclusionsOur results provide the first direct evidence of a causal role of caspase 3 activation in the cellular changes during kainic acid-mediated excitotoxicity. These findings may highlight novel pharmacological strategies to arrest disease progression and control seizures that are refractory to classical anticonvulsant treatment.

PARP cleavage, DNA fragmentation, and pyknosis during excitotoxin-induced neuronal death

Poly(ADP-ribose)polymerase-1 (PARP-1) is a nuclear enzyme activated by DNA breaks and serves a role in DNA repair through the formation of polymers (poly(ADP)ribosylation) at sites of DNA damage. PARP-1 is activated by DNA damage in neurons of the hippocampus and cerebral cortex following excessive exposure to glutamate receptor agonists such as NMDA or kainic acid. In addition, recent studies suggest that degradation of PARP-1 occurs in cells that undergo apoptotic versus nonapoptotic forms of cell death. To investigate this process further, we examined the spatiotemporal aspects of excitotoxic injury in the rodent visual cortex by making focal intracerebral injections of kainic acid. These injections resulted in DNA damage, PARP-1 activation, and neuronal cell death over a 5-day period. Rapid neuronal cell injury assessed by Fluoro-Jade staining appeared within hours, but increased TUNEL staining occurred only after 24 h. A dramatic increase in caspase-3 activity, as well as an increase in the number of neurons containing active caspase-3, peaked 2 days after injury. Last, increased PARP-1 immunoreactivity and PARP-1 cleavage reached peak levels 2 to 3 days after delivering the excitotoxin. These findings suggest that increased caspase-3 activity may regulate the degradation of PARP-1 in subsets of cortical neurons during excitotoxic cell death.

In situ transblot and immunocytochemical comparisons of astrocytic connexin-43 responses to NMDA and kainic acid in rat brain

Intracerebral injection of kainic acid (KA) in rat brain was previously found to cause altered immunohistochemical recognition of connexin-43 (Cx43) epitopes (epitope masking) with different sequence-specific antibodies against this gap junction protein. We demonstrate here that similar alterations occur when nitrocellulose membranes containing protein transferred from fresh cryostat sections of KA-injected brain are probed with these antibodies (in situ transblotting), indicating that epitope masking is not a result of epitope alteration due to fixation conditions used in earlier studies. Alterations in immuno-recognition of astrocytic Cx43 subsequent to injections of NMDA were also observed and were similar to those seen with KA in some, but not all respects. The results provide further indications of Cx43 molecular modification in excitotoxin-lesioned tissue and suggest that the sequelae of reactions by astrocytes and their gap junctions in these tissues is dependent on cell-type susceptibility to excitotoxin action.

Antagonists of the NMDA receptor and allopurinol protect the olfactory cortex but not the striatum after intra-cerebral injection of kainic acid

Overstimulation of the NMDA receptor, as well as generation of excessive amounts of free radicals, has been implicated in excitotoxic brain injuries. We report here that two antagonists of the NMDA receptor and an inhibitor of the free radical-generating enzyme, xanthine oxidase, protect the olfactory cortex but not the striatum after intrastriatal injection of kainic acid. Our results suggest the existence of a precise link between excitotoxic activation of the NMDA receptor and neuropathology related to excessive amounts of free radicals. The focal point of this link may be the entry of Ca 2+ through the NMDA receptor and the consequent activation of proteases and free radical-generating systems.

Autoradiographic analysis of [ 3H]ryanodine binding sites in rat brain: Regional distribution and the effects of lesions on sites in the hippocampus

Quantitative and qualitative autoradiographic methods together with lesion approaches were used to determine the distribution of [ 3H]ryanodine binding sites in rat brain and the neuronal localization of these sites in the hippocampus. In normal animals, levels of [ 3H]ryanodine binding sites ranged from a low of about 1 fmol/mg tissue in subcortical structures to a high of 12–18 fmol/mg tissue in subregions of the hippocampus and the olfactory bulb. Relatively high densities of sites (5–9 fmol/mg tissue) were also seen in the olfactory tubercle, most areas of the cerebral cortex, accumbens nucleus, striatum, lateral septal nuclei, pontine nucleus, superior colliculus and granule cell layer of the cerebellum. Specific binding was undetectable in white matter. In experimental animals, intracerebral injections of kainic acid caused neuronal degeneration and a near total depletion of [ 3H]ryanodine binding sites in the dentate gyrus and in fields CA1, CA2 and CA3 of the hippocampus. Injections of kainic acid that left dentate granule cells largely intact while destroying all neurons in field CA3 had no effect on binding sites in the dentate gyrus. However, these lesions substantially reduced the density of binding in field CA3, leaving a narrow band of sites outlining the position of the degenerated CA3 pyramidal cells. Mechanical knife-cut lesions that severed the granule cell mossy fiber input to field CA3 reduced the density of binding sites in the CA3 region. The results indicate that [ 3H]ryanodine binding sites in brain are heterogeneously distributed and suggest that a proportion of these sites in the hippocampus may be contained in mossy fiber terminals where a presumptive calcium channel/ryanodine receptor complex may be involved in the regulation of calcium mobilization and/or neurotransmitter release.

Phenoxybenzamine reduces mortality associated with intracerebral injections of excitatory neurotoxins

Intracerebral injections of kainic acid produce gross hematuria, renal cortical necrosis, and an associated high mortality. Hematuria is virtually eliminated and mortality significantly reduced by pretreatment with phenoxybenzamine.

Heat shock proteins as markers of neural injury

Systemic or intracerebral injections of kainic acid induced immunoreactivity for the 72 kDa heat shock protein (HSP72) in individual neurons of the rat brain in patterns matching the known histopathology of the particular injury. HSP72 immunostaining was also induced in and around areas of infarction following experimental strokes. These results suggest that HSP72 immunocytochemistry may be used as a marker of cellular injury in the mammalian brain.

In vivo and in vitro staining of acidophilic neurons as indicative of cell death following kainic acid-induced lesions in rat brain

An in vivo method for positively staining dead neurons was developed and compared with an in vitro staining method using acid fuchsin. Neurons previously killed by intracerebral injections of kainic acid were selectively stained by trypan blue within 15 min of its injection in vivo into the central nervous system of rats. Such staining persisted for at least 4 days in vivo, and there was no evidence that trypan blue itself was toxic to the remaining cells. Intense staining of neurons with acid fuchsin in vitro was first observed in brain sections of rats killed about 6h after kainic acid injection. This time was approximately 2-3 h before trypan blue, in vivo, was able to stain neurons. Thus, the loss of transport mechanisms (at least for trypan blue) apparently occurs subsequent to the development of basic products stainable with acidic dyes. At the earliest times, acid fuchsin stained neurons which had not yet lost Nissl substance, whereas the majority of trypan blue-stained neurons were not stained with Nissl dyes. After 24 h the majority of neurons stained with either acid fuchsin or trypan blue were Nissl-stain negative. The combination of staining with trypan blue in vivo with subsequent counterstaining of brain sections with acid fuchsin in vitro may have a potential use in the determination of the time of neuronal death in vivo.

Imaging of the degeneration of neurons and their processes in rat or cat brain by 45CaCl2 autoradiography or 55CoCl2 positron emission tomography.

The possibility of using radiolabeled divalent cations to visualize nerve cell degeneration in the brain was investigated after intoxication with neurotoxins. At different survival times after the intracerebral injection of kainic acid or 6-hydroxydopamine, autoradiographs were made from brain sections of rats that had received 45CaCl2 intravenously 24 h before death. Brain sections, adjacent to those used for autoradiography, of the 6-hydroxydopamine-treated rats were used for histofluorescence of catecholamines to check the neurochemical effect of the treatment. These experiments show that radioactive Ca accumulates in brain tissue during a particular phase of degeneration. Not only could degenerating cell bodies be traced by 45Ca autoradiography, but also degenerating nerve terminals in the striato-nigral and nigro-striatal projection systems. In positron emission tomography (PET) studies, 55CoCl2 was used as a marker for Ca2+. Unilateral lesions of the cat forebrain, produced by kainic acid, could be imaged in vivo by PET with 55CoCl2. PET with this radiolabel may provide diagnostic potentials for human neurodegenerative disorders.

Possible destruction of cholinoceptive neurons by overstimulation of cholinergic tracts.

It is well established that intracerebral injections of kainic acid may cause not only neuronal cell destruction at the injection site, but also losses in some distant regions. The mechanisms are different. The distant, but not the local, destruction can be produced by folic as well as by kainic acid and prevented by pretreatment of the animal with diazepam. Overexcitation of excitatory projections is believed responsible for the distant damage and evidence is presented that in some instances the projections involved are cholinergic. Thus, for example, injections of kainic acid or folic acid into the substantia innominata of rats destroy neurons in areas such as the pyriform cortex and amygdala which receive cholinergic projections from the injected area. Some of the destroyed neurons are GABAergic. That the distant toxicity in these areas can be partially blocked by scopolamine and is accompanied by decreases in the number of muscarinic binding sites is consistent with a cholinergic mechanism. Distant damage also occurs in the thalamus but this appears to be mediated by a noncholinergic projection. Similar injections of folic acid or kainic acid into the rostral pontine tegmentum, another area with cholinergic cells, cause destruction of both dopaminergic and GABAergic neurons in the substantia nigra. The effect on the GABAergic but not that on the dopaminergic cells is blocked by scopolamine. The results are discussed in relation to possible mechanisms of epilepsy and of selective neuronal losses in diseases such as Parkinson's disease.

Long-term local and distal increases in tryptophan hydroxylase activity following intracerebral kainic acid injections in the rat

The administration of kainic acid (1–2 μg) into the right striatum of adult rats resulted in a marked local increase in tryptophan hydroxylase activity (+ 54–106%). This change was significant as soon as on the second day after the treatment and persisted for at least 12 days. In addition, long-lasting elevations of tryptophan hydroxylase activity were also observed in the anterior raphe area, septum and ipsilateral hippocampus and cerebral cortex. In contrast, the intrahippocampal injection of kainic acid (1 μg) induced a long-term increase in tryptophan hydroxylase activity only in the injected structure. In all cases, the changes in tryptophan hydroxylase activity were associated with significant increases in the V max of the enzyme with no alteration of its apparent affinities for tryptophan and the pterin cofactor. Studies of the sensitivity of tryptophan hydroxylase from control and from kainic acid-treated rats to in vitro activating conditions (Ca +-dependent phosphorylation, partial trypsinization, exposure to sodium dodecyl sulfate) suggest that the intrastriatal injection of the neurotoxin induced a long-lasting activation of the enzyme. These findings indicate that intracerebral injections of kainic acid may be a valuable approach to explore further the mechanisms controlling tryptophan hydroxylase activity in vivo.

Necrotizing effects of kainic acid on neurons in the pigeon brain: Histological observations

The neurotoxin kainic acid (KA) has been shown to destroy neurons in the pigeon paleostriatal complex (PC), the avian analogue of the caudato-putamen and globus pallidus. In this earlier study the movement disorders in pigeons were strikingly similar to those reported by others in rats following intracerebral injection of KA into the corpus striatum. The toxic influences of KA on other parts of the pigeon brain have not been described. Therefore, KA was injected into areas of the telencephalon, diencephalon, mesencephalon and cerebellum. Areas sensitive to KA showed a marked cell loss and the neuropil exhibited spaces that contained fragments of necrotic neurons. The injection sites were invaded by glia and granulocytes. Kainic acid had a local necrotizing effect; for example, it destroyed neurons in the PC, nucleus rotundus, nucleus spiriformis lateralis, nucleus ruber and neurons of the cerebellar cortex. An apparent long-distance effect of KA was also observed, since intracerebral injections of KA into the PC was followed by cell loss in the ipsilateral nucleus of the ansa lenticularis. Kainic acid has proved to be a potent neurotoxin with a pronounced necrotizing effect upon neurons of the pigeon brain.

Spontaneously recurrent seizures after intracerebral injections of kainic acid in rat: a possible model of human temporal lobe epilepsy

Intrastriatal injections of kainic acid in rats acutely induced repeated episodes of clonic convulsions. Spontaneously recurrent generalized seizures and a potentiation of the convulsant effects of pentylenetetrazol were then observed in most of the injected rats several weeks after surgery. In addition to marked loss of striatal neurons, limbic pathological alterations similar to those found in human temporal lobe epilepsy were observed in the brains of the kainic-acid treated rats. It is proposed that this preparation might serve as an animal model of human temporal lobe epilepsy.

Widespread patterns of neuronal damage following systemic or intracerebral injections of kainic acid: A histological study

The effect of systemic and intracerebral injections of kainic acid, a potent neuroexcitatory and neurotoxic analogue of glutamate, has been studied in the rat using a variety of light- and electron-microscopic techniques. Systemic injections of 12 mg/kg produce a high incidence of ‘wet dog shakes’ and convulsions; seizure activity is consistently associated with neuronal damage. The initial neuropathological reactions include dendritic and glial dilations in discrete areas of the neuropil; at this time and subsequently, affected neuronal somata either appear swollen and pale, or are shrunken with dark cytoplasm. In the most severely affected areas, the lesion progresses to severe disruption of the neuropil. A vvidespread pattern of damage is seen throughout the forebrain, which is relatively consistent from brain to brain and is characterized by the parallel degeneration of pairs or groups of structures with extensive axonal interconnections. The commonly affected areas include the olfactory cortex, amygdaloid complex, hippocampus, and related parts of the thalamus and neocortex. Intracerebral injections of 2–6 nmol produce extensive neuronal damage in distant structures, as well as at the injection site. The pattern of distant damage varies with the site of the injection and appears to reflect axonal connections between the affected areas near the injection and the distant areas of damage. For example, injections into the olfactory cortex tend to produce damage in the amygdala, the mediodorsal thalamic nucleus, and related areas of the frontal cortex, as well as in the olfactory cortex itself. Injections into the posterior part of the olfactory cortex which involve the entorhinal cortex tend to produce severe degeneration in field CA 1 of the hippocampus, although field CA 3 is more severely damaged following intraventricular, intrahippocampal or intrastriatal injections. These observations suggest that kainic acid may exert at least part of its toxic effects via axonal connections. Furthermore, the widespread damage induced by intracerebral injections indicates that caution must be exercised in the use of kainic acid to produce circumscribed lesions, and that careful histological evaluation of such lesions is required.

Changes of hippocampal Met-enkephalin content after recurrent motor seizures.

Kainic acid (KA), a powerful neurotoxic analogue of glutamate, has been extensively used as a tool for selectively lesioning neuronal cell bodies; however, axons or nerve terminals are spared from damage in the area injected with kainic acid. Injections of this neurotoxin in various brain regions were successfully used to locate cell bodies of neurones containing substance P, enkephalin and other putative neurotransmitters. While attempting to locate the cell bodies of the enkephalin containing neurones present in hippocampus using KA injections, we found that a few days after intracerebral injections of KA a drastic increase in the Met-enkephalin (ME) content of hippocampus occurs. We now describe the delayed increase in hippocampal ME content elicited by intracerebral KA injections and examine the possible mechanism that is operative in causing this increase. Moreover, we provide some evidence suggesting that the increase in ME content elicited by intracerebral injections of KA may be related to the recurrent motor seizures elicited by intracerebral injections of KA.

Local and distant neuronal degeneration following intrastriatal injection of kainic acid.

After intrastriatal injection, the neurotoxin, kainic acid, was cleared from the rat forebrain in a biphasic manner with 70% eliminated within 2 hours; by 24 hours after infusion, less than 1% of the kainic acid remained in the forebrain. The kainic acid diffused into adjacent brain structures, achieving mu molar concentrations in several regions ipsilateral to the injected striatum. At various times after intrastriatal injection of 9.3 nmoles of kainic acid, the brain was serially sectioned; the sections were stained for Nissl substance with cresyl violet or for degenerating neurons with the ammoniacal silver method. Neuronal degeneration spread unevenly into contiguous structures from the central sphere in the injected striatum and affected the ipsilateral pyriform cortex and amygdala, the deep layers of the overlying cerebral cortex, and the medial aspects of the bed nucleus of the stria terminalis and of the nucleus accumbens. In half of the rats, the pyriform cortex contralateral to the side of injection also underwent degeneration. A subpopulation of pyramidal cells in layer IV of the lateral neocortex and the CA3-CA4 pyramidal cells in the ipsilateral hippocampus were selectively affected, whereas adjacent neurons remained intact. The distribution of agyrophilic fibers and terminals in subcortical structures was consistent with the degeneration of neurons of origin in the affected striatal and extrastriatal regions. Brain sections stained by the gold sublimate technique from rats perfused 20 days after injection revealed an intense astrocytic response in all areas affected by acute neuronal degeneration. Extrastriatal damage could be markedly reduced by injection of lower doses of kainic acid (2.3 nmoles) with brief anesthesia; under these conditions, however, the subpopulation of large striatal neurons were relatively resistant, as compared to the Golgi II neurons. These studies demonstrate significant and variable neuronal degeneration beyond the primary site of the lesion after intracerebral injection of kainic acid; several factors affect the pattern of degeneration, including the amount of kainic acid injected, its biological activity, its diffusion, duration of anesthesia, and variable sensitivity of neurons. Consequently, care must be exercised in the use of this neurotoxin to determine the extent and selectivity of neuronal damage, particularly with reference to neuronal vulnerability beyond the central sphere of intrinsic neuronal degeneration.

Thalamic injections of kainic acid produce myocardial necrosis

Bilateral injections of 2–5 nmol of kainic acid into the thalamus produce periarteriorlar myocardial necrosis. In addition, gross hematuria is usually observed. Electrolytic lesions of the same area of brain and intracerebral injections of kainic acid at several other locations fail to produce these peripheral changes. Kainic acid at much higher doses subcutaneously or intraperitoneally is also inactive. Urinary noradrenaline levels are increased up to 10-fold during the post-injection period. Some protection against myocardial damage may be produced by reserpine or 6-hydroxydopamine, but atropine seems to confer no protection. The fact that myocardial damage may result from intracerebral lesions and/or pathological stimulation by kainic acid may have clinical implications. Cardiac damage occasionally results in humans from strokes and intracerebral hemorrhage and no satisfactory explanation has ever been offered for the phenomenon of interstitial myocardial fibrosis. The kainic acid model may be one means of studying this phenomenon.

The use of kainic acid in the localization of enzymes in the substantia nigra

The selective destruction of neuronal perikarya via intracerebral injections of kainic acid was used to elucidate the cellular location of four neurotransmitter-related enzymes in the substantia nigra (SN). Two weeks after intranigral injections of kainic acid, dopamine-sensitive adenylate cyclase, glutamic acid decar☐ylase (GAD), choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were measured in the SN. Histological examination of the SN, and a reduction of striatal tyrosine hydroxylase (TH) activity by 94%, confirmed the extensive loss of neuronal cell bodies in the SN. Dopamine stimulation of adenylate cyclase was not reduced in the lesioned SN, supporting the view that dendritically-released dopamine can regulate cyclic AMP synthesis in afferent terminals to these dendrites. Nigral GAD activity was significantly reduced by the lesions, suggesting that there are GAD-containing perikarya in the SN. CAT activity was not affected by the kainic injections, indicating the absence of cholinergic perikarya in the SN. Nigral AChE activity was significantly decreased after kainic injections, thus confirming the presence of AChE within the nigral perikarya. The results suggest that dopamine-sensitive adenylate cyclase and CAT are located within afferents to the SN, while GAD and AChE are found, to some extent at least, in neuronal soma of the SN. The differential effects of kainic acid on these enzymes suggest that this compound may be a useful neurochemical tool with which to determine the cellular distribution of enzyme systems in the central nervous system.

Neurochemical sequelae of kainate injections in corpus striatum and substantia nigra of the rat

Unilateral injection of 2 μg kainic acid into the substantia nigra of the rat results in a 45% decrease in tyrosine hydroxylase activity in the injected substantia nigra and in the ipsilateral corpus striatum. In contrast, the GABAergic nerve terminals in the substantia nigra are unaffected by this treatment. Injection of kainic acid into the striatum results in a 60% decrement in the activity of glutamate decarboxylase and of endogenous GABA levels in the ipsilateral substantia nigra whereas tyrosine hydroxylase activity remains unchanged; in addition, dopamine-sensitive adenylate cyclase activity in the ipsilateral substantia nigra decreases by 74%. These findings further support the hypothesis that intracerebral injections of kainic acid cause degeneration of neurons with cell bodies near the injection site while sparing axons passing through or terminating in the region.