Karuppagounder S. Saravanan

Acute intranigral infusion of rotenone in rats causes progressive biochemical lesions in the striatum similar to Parkinson's disease

We examined in Sprague-Dawley rats whether intranigral administration of complex-I inhibitor, rotenone, produces biochemical lesions in the striatum similar to those observed in Parkinsons disease (PD). Unilateral stereotaxic infusion of rotenone (2-12 mug in 1 mul) into substantia nigra (SN) pars compacta caused significant inhibition of complex-I activity and increased production of hydroxyl radicals in vivo as measured employing spectrophotometric and HPLC-electrochemical procedures, respectively. It also caused a significant time- and dose-dependent reduction of dopamine level, but not serotonin, in the ipsilateral striatum when assayed using an HPLC electrochemical method. This effect was found to be progressive for 90 days. A dose-dependent decrease in nigral glutathione level, as measured fluorimetrically, was also observed to be progressive till 90th day. A significant decrease in tyrosine hydroxylase immunoreactivity in the striatum (73 +/- 8.4% as assessed by densitometric studies) or in SN ipsilateral to the side of infusion suggested nigrostriatal neuronal degeneration. A dose of rotenone (6 microg in 1 microl) that caused 55% striatal dopamine depletion when infused into the SN failed to affect serotonin levels in the terminal regions when infused into the nucleus raphe dorsalis, indicating rotenones specificity of action towards dopaminergic neurons. Our findings suggest that unilateral infusion of rotenone reproduces neurochemical and neuropathological features of hemiparkinsonism in rats and indicate an active involvement of oxidative stress in rotenone-induced nigrostriatal neurodegeneration. The present study also demonstrates more sensitivity of dopaminergic neurons towards rotenone and establishes mitochondrial complex-I damage as one of the major contributory components of neurodegeneration in PD. The progressive nature of pathology in this model closely mimics idiopathic PD, and absence of mortality warrants the use of this model in drug discovery programs.

Non-steroidal anti-inflammatory drug sodium salicylate, but not diclofenac or celecoxib, protects against 1-methyl-4-phenyl pyridinium-induced dopaminergic neurotoxicity in rats

We evaluated the hydroxyl radical (*OH) scavenging action of nonsteroidal anti-inflammatory drugs (NSAIDs), sodium salicylate (SA), diclofenac and celecoxib in Fentons reaction and their neuroprotective effects in 1-methyl-4-phenylpyridinium (MPP(+))-induced striatal dopamine (DA) depletion in rats. Salicylate hydroxylation procedure employing HPLC-electrochemistry was used to assay formation of *OH in Fentons reaction in test tubes. While SA dose- and time-dependently hydroxylated itself and inactivated *OH, celecoxib (up to 10 mM) showed no effect on *OH formation and diclofenac caused a reduction in *OH generation only at high doses (100 microM-10 mM). Administration of the non-selective cyclooxygenase (COX) inhibitor, SA (50, 100 mg/kg, i.p.) significantly attenuated striatal DA depletion caused by intrastriatal infusion of MPP(+) (100 nmol in 4 microl). Treatment with another nonselective, reversible COX inhibitor, diclofenac (5, 10 mg/kg) did not protect against MPP(+)-induced DA depletion. The selective COX-2 inhibitor, celecoxib (2.5-50 mg/kg) treatment exacerbated MPP(+)-induced decrease in DA. Failure of celecoxib or diclofenac to render protection in animals against MPP(+)-induced DA depletion indicates absence of prostaglandin involvement in MPP(+) action. These results also suggest that the neuroprotective ability of SA is independent of prostaglandin mediation. A relationship between inactivation of *OH by SA and its ability to protect DA depletion in the striatum caused by MPP(+) indicates a direct involvement of *OH in the action of this neurotoxin. The present study establishes potent neuroprotective activity of SA and suggests the use of aspirin in adjuvant therapy in Parkinsons disease.

Melatonin protects against rotenone-induced oxidative stress in a hemiparkinsonian rat model.

Abstract:  In the present study, we evaluated the effect of melatonin, a well‐known free radical scavenger and neuroprotector, against rotenone‐induced oxidative stress in a hemiparkinsonian rat model. The effect of melatonin on glutathione (GSH) depletion caused by unilateral, intranigral infusion of rotenone was investigated employing a spectrofluorimetric procedure. We also studied the effect of melatonin on rotenone‐induced changes in the antioxidant enzymes superoxide dismutase (SOD) and catalase in the cytosolic fractions of substantia nigra (SN), employing spectrophotometric procedures. Rotenone‐induced hydroxyl radicals (•OH) in the isolated mitochondria, as measured employing a sensitive HPLC‐electrochemical method, were significantly scavenged by melatonin. Melatonin treatment restored the rotenone‐induced decrease in GSH level and changes in antioxidant enzyme (SOD and catalase) activities in the SN. Our results strongly indicate melatonins beneficial use in Parkinsons disease therapy as an antioxidant.

Acetaminophen and aspirin inhibit superoxide anion generation and lipid peroxidation, and protect against 1-methyl-4-phenyl pyridinium-induced dopaminergic neurotoxicity in rats

We assessed the antioxidant activity of non-narcotic analgesics, acetaminophen and aspirin in rat brain homogenates and neuroprotective effects in vivo in rats intranigrally treated with 1-methyl-4-phenyl pyridinium (MPP+). Both drugs inhibited cyanide-induced superoxide anion generation, as well as lipid peroxidation in rat brain homogenates, the combination of the agents resulting in a potentiation of this effect. Acetaminophen or aspirin when administered alone or in combination, did not alter dopamine (DA) levels in the forebrain or in the striatum. Intranigral infusion of MPP+ in rats caused severe depletion of striatal DA levels in the ipsilateral striatum in rats by the third day. Systemic post-treatment of acetaminophen afforded partial protection, whereas similar treatment of aspirin resulted in complete blockade of MPP+-induced striatal DA depletion. While these findings suggest usefulness of non-narcotic analgesics in neuroprotective therapy in neurodegenerative diseases, aspirin appears to be a potential candidate in prophylactic as well as in adjuvant therapy in Parkinsons disease.

Behavioral differences in a rotenone-induced hemiparkinsonian rat model developed following intranigral or median forebrain bundle infusion

A mitochondrial complex-I inhibitor, rotenone was unilaterally infused into the substantia nigra pars compacta (SNpc) or median forebrain bundle (MFB) to create hemiparkinsonian animal models and investigated spontaneous and drug-induced stereotypic rotations, as well as certain postural behaviors in Sprague-Dawley rats. Animals infused intranigrally, but not intra-MFB, with rotenone exhibited spontaneous contralateral rotations immediately after recovery from anesthesia. Head position bias and elevated body swing test showed insignificant contralateral bias in animals with nigral damage but a significant ipsilateral bias in MFB-lesioned rats. General motor activity of the animals was reduced in both the groups as indicated by reduced performance on a Plus-Maze. Intranigrally, rotenone-infused animals exhibited progressive ipsilateral rotations when challenged with d-amphetamine on the 7th, 14th, 21st, and 28th days or with apomorphine on 9th, 16th, 23rd, and 30th days. However, animals that received rotenone in MFB exhibited ipsilateral or contralateral rotations when challenged respectively with d-amphetamine or apomorphine only in the 5th week (28th and 30th days). Stereotaxic administration of rotenone into SNpc or MFB caused a significant loss of dopamine in the ipsilateral striatum (>80% in SNpc; >95% in MFB), when assayed employing an HPLC equipped with electrochemical detector on the 32nd day. Neuronal loss in SNpc was confirmed in coronal sections stained with cresyl violet and revealed extension of lesion towards SN pars reticulata, in SNpc-infused animals. Our results demonstrate that rotenone-induced neurodegeneration is a slow, yet progressive process similar to that in idiopathic Parkinsons disease and unlike that observed in other classical neurotoxin-mediated lesions which are abrupt and developed in few hours to days. Thus, intranigral or intra-MFB infusion of rotenone could be used for producing hemiparkinsonian animal models in rats. These findings further suggest that, while both d-amphetamine and apomorphine-induced stereotypic rotations could be used as a valuable behavioral assay procedure to test novel drugs against Parkinsons disease, yet apomorpine-induced contralateral bias in turning is a reliable indicator of specific destruction in nigrostriatal pathway and development of postsynaptic dopamine receptor supersensitivity.

L-deprenyl protects against rotenone-induced, oxidative stress-mediated dopaminergic neurodegeneration in rats.

The present study investigated oxidative damage and neuroprotective effect of the antiparkinsonian drug, L-deprenyl in neuronal death produced by intranigral infusion of a potent mitochondrial complex-I inhibitor, rotenone in rats. Unilateral stereotaxic intranigral infusion of rotenone caused significant decrease of striatal dopamine levels as measured employing HPLC-electrochemistry, and loss of tyrosine hydroxylase immunoreactivity in the perikarya of ipsilateral substantia nigra (SN) neurons and their terminals in the striatum. Rotenone-induced increases in the salicylate hydroxylation products, 2,3- and 2,5-dihydroxybenzoic acid indicators of hydroxyl radials in mitochondrial P2 fraction were dose-dependently attenuated by L-deprenyl. L-deprenyl (0.1-10mg/kg; i.p.) treatment dose-dependently attenuated rotenone-induced reductions in complex-I activity and glutathione (GSH) levels in the SN, tyrosine hydroxylase immunoreactivity in the striatum or SN as well as striatal dopamine. Amphetamine-induced stereotypic rotations in these rats were also significantly inhibited by deprenyl administration. The rotenone-induced elevated activities of cytosolic antioxidant enzymes superoxide dismutase and catalase showed further significant increase following L-deprenyl. Our findings suggest that unilateral intranigral infusion of rotenone reproduces neurochemical, neuropathological and behavioral features of PD in rats and L-deprenyl can rescue the dopaminergic neurons from rotenone-mediated neurodegeneration in them. These results not only establish oxidative stress as one of the major causative factors underlying dopaminergic neurodegeneration as observed in Parkinsons disease, but also support the view that deprenyl is a potent free radical scavenger and an antioxidant.

Rats with unilateral median forebrain bundle, but not striatal or nigral, lesions by the neurotoxins MPP+ or rotenone display differential sensitivity to amphetamine and apomorphine

Rotenone and 1-methyl-4-phenyl pyridinium (MPP+) are two mitochondrial neurotoxins known to produce Parkinsons disease (PD) in experimental animals. In the present study, we compared drug-induced rotational asymmetry in rats lesioned using these neurotoxins at three distinct basal ganglia sites, the striatum, substantia nigra pars compacta (SNpc) and median forebrain bundle (MFB). The levels of dopamine (DA) in the ipsilateral striata of these hemiparkinsonian animals were assayed employing an HPLC-electrochemical procedure 2 days after the final rotational study. Rats infused with rotenone or MPP+ into the SNpc, but not into the striatum or MFB, exhibited contralateral rotations immediately after recovery from anesthesia. Irrespective of the lesion site or the toxin used, all the animals exhibited ipsilateral rotations when challenged with D-amphetamine. Apomorphine administration caused contralateral circling behavior in MFB-lesioned animals, but ipsilateral rotations in rats that received rotenone or MPP+ in the striatum or SNpc. Stereotaxic administration of rotenone into the MFB, SNpc or striatum caused a significant loss of DA in the ipsilateral striatum to varying degrees (96%, 62% and 30%, respectively, as compared to the contralateral side). However, unilateral MPP+ administration into the MFB, SNpc or striatum caused respectively about 98%, 74% and 59% loss of striatal DA. Behavioural observations and the neurochemical results indicate that, among the three anatomically distinct loci-lesioned, MFB-lesioned animals mimicked behavioral aberrations similar to nigral lesions caused by 6-hydroxydopamine, a classical parkinsonian neurotoxin. Moreover, the results point out that while both d-amphetamine and apomorphine-induced rotations could be considered as valuable behavioral indices to test novel drugs against PD, yet apomorphine-induced contralateral bias proves to be a more reliable indicator of specific destruction in the nigrostriatal pathway and development of post-synaptic DA receptor supersensitivity.

Serotonin synthesis inhibition in olivo-cerebellar system attenuates harmaline-induced tremor in Swiss albino mice

Recent experimental evidences point to the active role of central serotonin (5-HT) elicited mechanisms in the pathogenesis of tremor. The present study was undertaken to investigate the effects of p-chlorophenylalanine (pCPA), a specific tryptophan hydroxylase inhibitor and a central 5-HT depletor, on the neurochemical processes that occur synchronously in olivary nucleus (ON) and cerebellum during harmaline-induced tremor in mice. Tremor appeared by 3-4 min following harmaline administration, and reached its peak by 25 min for the doses (10-30 mg/kg) studied. Peak of harmaline-tremor coincided with increases in 5-HT in ON and cerebellum, as assayed employing HPLC-electrochemistry. Administration of pCPA caused significant depletion in 5-HT level in both the regions analyzed, and also significantly inhibited harmaline-induced tremor. Our present results support the earlier electrophysiological evidences that harmaline-induced tremor originates from ON, and confirm the role of 5-HT in the genesis of this motor neuronal dysfunction.

Unilateral implantation of dopamine-loaded biodegradable hydrogel in the striatum attenuates motor abnormalities in the 6-hydroxydopamine model of hemi-parkinsonism

Dopaminergic functional recovery following controlled release of dopamine from biodegradable polymer matrices implanted in the lesioned striatum was investigated in a hemiparkinsonian animal model. Significant dopamine depletion in the striatum ipsilateral to the side of infusion was observed in animals unilaterally infused with 6-hydroxydopamine (6-OHDA) in the substatia nigra. These animals displayed apomorphine-induced contralateral rotational behavior, when examined on the 16th day. Implantation of a controlled release delivery system (hydrogel obtained by mixing dextran dialdehyde cross-linked with gelatin) containing dopamine in the denervated striatum on the 1st day or the 18th day significantly abolished the apomorphine-induced contralateral rotational behavior in these animals. The recovery was visible for about 17 days, thereafter the behavioral bias reappeared. The present results indicate that dopamine released from the polymer matrices alleviates behavioral bias in experimental parkinsonism, implying use of such technologies as an alternative method for the treatment of Parkinsons disease. This approach is useful in reducing the oral dose of drugs that are with severe systemic effects, and that develop tolerance.

Apoptotic Mode of Cell Death in Substantia Nigra Following Intranigral Infusion of the Parkinsonian Neurotoxin, MPP+ in Sprague-Dawley Rats: Cellular, Molecular and Ultrastructural Evidences

The potent parkinsonian neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) is known to cause dopaminergic neurodegeneration in nigrostriatal system. In the present study we investigated the nuclear morphology of cells in the substantia nigra pars compacta (SNpc) region of rats following unilateral intranigral infusion of the active metabolite, 1-methyl-4-phenyl pyridinium ion (MPP+), which resulted in a dose-dependent and prolonged dopamine depletion in the ipsilateral striatum. There appeared a substantial loss of tyrosine hydroxylase immunoreactive neurons in the SNpc that received the neurotoxin. Specific nuclear staining with Hoechst 33342 or acridine orange revealed bright pyknotic, shrunken, distorted nuclei and condensed chromatin with perinuclear nucleolus respectively following visualization with the former and latter dyes in the ipsilateral SNpc, as compared to the round, intact nuclei and centrally positioned nucleolus in the contralateral side. Ultrastructural details of the nucleus under transmission electron microscope confirmed distorted nuclear organization with shrunken or condensed nuclei and disrupted nuclear membrane. These features are typical of nucleus undergoing apoptosis, and suggest that MPP+ causes dopaminergic neuronal death through an apoptotic mode. Typical laddering pattern of genomic DNA isolated from the ipsilateral SN in agarose gel electrophoresis conclusively established apoptosis following intranigral administration of MPP+ in rats.