Neuroprotective effect of pyruvate and oxaloacetate during pilocarpine induced status epilepticus in rats

Recent research data have shown that systemic administration of pyruvate and oxaloacetate causes an increased brain-to-blood glutamate efflux. Since increased release of glutamate during epileptic seizures can lead to excitotoxicity and neuronal cell death, we tested the hypothesis that glutamate scavenging mediated by pyruvate and oxaloacetate systemic administration could have a neuroprotective effect in rats subjected to status epilepticus (SE). SE was induced by a single dose of pilocarpine (350mg/kgi.p.). Thirty minutes after SE onset, a single dose of pyruvate (250mg/kgi.p.), oxaloacetate (1.4mg/kgi.p.), or both substances was administrated. Acute neuronal loss in hippocampal regions CA1 and hilus was quantitatively determined five hours after SE onset, using the optical fractionator method for stereological cell counting. Apoptotic cascade in the hippocampus was also investigated seven days after SE using caspase-1 and -3 activity assays. SE-induced neuronal loss in CA1 was completely prevented in rats treated with pyruvate plus oxaloacetate. The SE-induced caspase-1 activation was significantly reduced when rats were treated with oxaloacetate or pyruvate plus oxaloacetate. The treatment with pyruvate and oxaloacetate caused a neuroprotective effect in rats subjected to pilocarpine-induced SE.     

Depression and/or potentiation of cortical responses after status epilepticus in immature rats

Lithium-pilocarpine status epilepticus (SE) resulted in delayed changes of single cortical interhemisperic (transcallosal) responses in immature rats. Low-frequency stimulation inducing depression and/or potentiation was studied to analyze possible dynamic changes in cortical responses. Status was elicited in 12-day-old (SE12) or 25-day-old (SE25) rats. Control siblings received saline instead of pilocarpine. Interhemispheric responses were elicited by stimulation of the sensorimotor region of the cerebral cortex 3, 6, 9, 13, or 26 days after status. A series of 5 biphasic pulses with intensity equal to twofold threshold were used for stimulation. The interval between pulses was 100, 125, 160, 200 or 300 ms, eight responses were always averaged. Peak amplitude of the first positive, first negative and second positive waves was measured and responses to the second, third, fourth and fifth pulse were compared with the first one. Animals after status epilepticus as well as lithium-paraldehyde controls exhibit a frequency depression at nearly all the intervals studied. An outlined increase of responses in SE rats in comparison with the controls three days after SE stayed just below the level of statistical significance. In addition, animals in the SE12 group exhibited potentiation of responses at this interval after SE. With longer intervals after SE, the relation between SE and control animals changed twice resulting in a tendency to lower amplitude of responses in SE than in control rats 26 days after SE. Rats in the SE25 group exhibited higher responses than controls 13 days after status, but this difference was not present at the longest interval after SE. Low-frequency stimulation did not reveal increased cortical excitability as a long-lasting consequence of status epilepticus induced in immature rats. In addition, the outlined differences between SE and control rats changed with the time after SE.     

Effect of alpha-phenyl-N-tert-butylnitrone on diabetes and lipid peroxidation in BB rats.

Oxygen free radicals have been shown to interfere with pancreatic islet beta cell function and integrity, and have been implicated in autoimmune type 1 diabetes. We hypothesized that the spontaneous autoimmune type 1 diabetes of the BB rat would be prevented by in vivo administration of a free-radical spin trap, alpha-phenyl-N-tert-butylnitrone (PBN). Twenty-eight diabetes-prone (BBdp) and 13 non-diabetes-prone (BBn) rats received PBN (10 mg/kg) subcutaneously twice daily, and 27 BBdp and 12 BBn rats received saline as controls. Rats were treated from age 47 +/- 6 days until diabetes onset or age 118 +/- 7 days. PBN caused no growth, biochemical, or hematological side effects. Sixteen control BBdp rats became diabetic (BBd, mean age 77 +/- 6 days) and six demonstrated impaired glucose tolerance (IGT rats). The incidence of diabetes and IGT was not different in PBN-treated BBdp rats. Saline-treated rats showed no differences in pancreatic malondialdehyde (MDA) contents of BBd, IGT rats, and the BBdp that did not develop diabetes, versus BBn rats (2.38 +/- 0.35 nmoL/g). Among rats receiving PBN, BBn had lower pancreatic MDA than BBd and IGT rats (1.38 +/- 0.15 vs. 1.88 +/- 0.15 and 2.02 +/- 0.24 nmoL/g, p < 0.05), but not than BBdp rats (1.78 +/- 0.12 nmoL/g, ns). BBn rats receiving PBN also had lower pancreatic MDA than the saline controls (p < 0.05). Thus, PBN is remarkably nontoxic and is able to decrease MDA in the absence of the autoimmune process, but does not prevent diabetes. A combination of PBN with other complementary antioxidant agents may hold better promise for disease prevention.     

Are acute changes after status epilepticus in immature rats persistent?

Early consequences of lithium-pilocarpine convulsive status epilepticus (SE) were studied six days after this status had been induced in rat pups at the age of either 12 or 25 days. Studies of spontaneous EEG activity demonstrated the presence of epileptic phenomena (isolated spikes) in both hippocampus and cortex (cortical spikes were more expressed in the older group). There were no marked behavioral correlates of spikes and transition into the ictal phase was exceptional. The motor performance on a rotorod and a horizontal bar was the same in experimental and control rats of both ages. Behavior in the open field was changed in a reverse manner in the two age groups: the locomotor activity of rats with induced seizures at the age of 12 days was significantly lower than that of their control siblings, whereas animals undergoing status at the age of 25 days were hyperactive. In addition, they also exhibited increased exploratory activity (rearing) and their habituation to the open field was deranged. Nissl-stained brain sections demonstrated extensive brain damage in the older group in contrast to the negative findings in younger animals. EEG, behavioral and morphological changes induced by status epilepticus in developing rats persisted for 6 days after the status. They markedly differed according to the age of animals.     

Antiepileptic and neuroprotective effects of human umbilical cord blood mononuclear cells in a pilocarpine-induced epilepsy model

Status epilepticus (SE) is a condition of persistent seizure that leads to brain damage and, frequently, to the establishment of chronic epilepsy. Cord blood is an important source of adult stem cells for the treatment of neurological disorders. The present study aimed to evaluate the effects of human umbilical cord blood mononuclear cells (HUCBC) transplanted into rats after induction of SE by the administration of lithium and pilocarpine chloride. Transplantation of HUCBC into epileptic rats protected against neuronal loss in the hippocampal subfields CA1, CA3 and in the hilus of the dentate gyrus, up to 300 days after SE induction. Moreover, transplanted rats had reduced frequency and duration of spontaneous recurrent seizures (SRS) 15, 120 and 300 days after the SE. Our study shows that HUCBC provide prominent antiepileptic and neuroprotective effects in the experimental model of epilepsy and reinforces that early interventions can protect the brain against the establishment of epilepsy.     

Thyroxine attenuates hippocampal neuronal damage caused by ischemia in the rat.

We investigated the effect of thyroxine against neuronal damage caused by ischemia in the rat. Neuronal damage was evaluated in the hippocampal CA1 subfield 7 days after a 10 min forebrain ischemia. Thyroxine was administered to animals divided in three groups: 15 min prior to ischemia (group 1), immediately after ischemia (group 2), and both before and after ischemia (group 3). The treatment of rats with a single dose of thyroxine given pre- or postischemia failed to prevent the loss of CA1 pyramidal cells. In contrast, repetitive administration of thyroxine before and after ischemia reduced the damage of the CA1 pyramidal cells. The mechanisms possibly underlying this neuroprotective effect are discussed.     

Nitric Oxide Participates in the Induction of Brain Ischemic Tolerance via Activating ERK1/2 Signaling Pathways

The present study was undertaken to observe in vivo changes of expression and phosphorylation of ERK1/2 proteins during brain ischemic preconditioning and effects of inhibiting generation of nitric oxide (NO) on the changes to determine the role of ERKs in the involvement of NO participating in the acquired tolerance. Fifty-five Wistar rats were used. Brain ischemic preconditioning was performed with four-vessel occlusion for 3 min. Total ERK1/2 proteins and phospho-ERK1/2 in the CA1 hippocampus were assayed with Western immunoblot. Total ERK1/2 proteins did not change in period from 5 min to 5 days of reperfusion after preconditioning stimulus. While the level of phospho-ERK1/2 increased obviously to 223, 237, 300, 385 and 254% of sham level at times of 5 min, 2 h, 1, 3 and 5 days after preconditioning stimulus, respectively (P < 0.01). Administration of L-NAME, an inhibitor of NO synthase, 30 min prior to preconditioning stimulus failed to induce change in total ERK1/2 proteins (P > 0.05). However, phospho-ERK1/2 increased only to 138 and 176% of sham level at 2 h and 3 days after preconditioning stimulus, respectively, when animals were pretreated with L-NAME. The magnitudes of the increase were obviously low compared with those (237 and 385%) in animals untreated with L-NAME at corresponding time points (P < 0.01), which indicated that phosphorylation of ERK1/2 normally induced by preconditioning stimulus was blocked apparently by administration of L-NAME. The results suggested that phosphorylation of ERK1/2, rather than synthesis of ERK1/2 proteins, was promoted in brain ischemic preconditioning, and that the promotion was partly mediated by NO signal pathway.     

Histaminergic H2 Action Protects Hippocampal CA1 Neurons by Prolonging the Onset of the Anoxic Depolarization in Gerbils

Abstract: The central histaminergic action on ischemia-induced neuronal damage was examined by evaluating the histological outcome and the direct current (DC) potential shift in the hippocampal CA1 region in gerbils. An intracerebroventricular administration of histamine (10–100 nmol) improved the delayed ischemic damage in hippocampal CA1 pyramidal cells produced by 3 min of transient forebrain ischemia. A high dose (75 nmol) of mepyramine, an H₁ antagonist, aggravated ischemia-induced neuronal damage, but not a low dose (0.75 nmol). Administration of cimetidine (4 nmol) and ranitidine (3 nmol), H₂ antagonists, aggravated the neuronal damage. An injection of histamine (100 nmol) prolonged the onset time of the ischemia-induced sudden shift in the extracellular DC potential (anoxic depolarization; AD) to 133% of that in control animals. Administration of mepyramine (75 nmol) did not markedly change the AD, whereas injections of cimetidine (40 nmol) and ranitidine (3 nmol) reduced the onset latency to 47 and 45%, respectively. These findings suggest that the central H₂ action serves to protect neurons by delaying the onset of AD in gerbils.     

Mannitol induces selective astroglial death in the CA1 region of the rat hippocampus following status epilepticus

In the present study, we addressed the question of whether treatment with mannitol, an osmotic diuretic, affects astrogliovascular responses to status epilepticus (SE). In saline-treated animals, astrocytes exhibited reactive astrogliosis in the CA1-3 regions 2-4 days after SE. In the mannitol-treated animals, a large astroglial empty zone was observed in the CA1 region 2 days after SE. This astroglial loss was unrelated to vasogenic edema formation. There was no difference in SE-induced neuronal loss between saline- and mannitol-treated animals. Furthermore, mannitol treatment did not affect astroglial loss and vasogenic edema formation in the dentate gyrus and the piriform cortex. These findings suggest that mannitol treatment induces selective astroglial loss in the CA1 region independent of vasogenic edema formation following SE. These findings support the hypothesis that the susceptibility of astrocytes to SE is most likely due to the distinctive heterogeneity of astrocytes independent of hemodynamics. [BMB Reports 2015; 48(9): 507-512]     

Free radical spin trap alpha-phenyl-N-tert-butyl-nitron inhibits caspase-3 activation and reduces brain damage following a severe forebrain ischemic injury.

It has been documented that alpha-phenyl-N-tert-butyl-nitron (PBN) possesses a potent neuroprotective effect when administered after transient focal cerebral ischemia. However, contradicting results were reported regarding its effect in transient global ischemia. To further elucidate the mechanism of PBN action, we have studied the effect of PBN on animal survival, histopathological outcome, and activation of caspase-3 following 30 min of global ischemia in vehicle- and PBN-treated rats. The results showed that 30 min of global ischemia was such a severe insult that no animal could survive beyond 2 d of reperfusion. Histopathological evaluation showed severe tissue edema and microinfarct foci in the neocortex and thalamus. Close to 100% damage was observed in the stratum and hippocampal CA1, CA3, and dentate gyrus subregions. Postischemic PBN treatment significantly enhanced animal survival and reduced damage in the neocortex, thalamus, and hippocampus. Immunohistochemistry demonstrated that caspase-3 was activated following ischemia in the striatum and the neocortex. PBN suppressed the activation of caspase-3 in both structures. It is concluded that PBN is a potent neuroprotectant against both focal and global ischemia; besides its function as a free radical scavenger, PBN may reduce ischemic brain damage by blocking cell death pathways that involve caspase-3 activation.     

Isoflurane but not halothane minimum alveolar concentration-sparing response of dexmedetomidine is enhanced in rats chronically treated with selective α2-adrenoceptor agonist

Halothane minimum alveolar concentration (MAC)-sparing response is preserved in rats rendered tolerant to the action of dexmedetomidine. It has been shown that halothane and isoflurane act at different sites to produce immobility. The authors studied whether there was any difference between halothane and isoflurane MAC-sparing effects of dexmedetomidine in rats after chronic administration of a low dose of this drug. Twenty-four female Wistar rats were randomly allocated into four groups of six animals: two groups received 10 μg/kg intraperitoneal dexmedetomidine for five days (treated groups) and the other two groups received intraperitoneal saline solution for five days (naive groups) prior to halothane or isoflurane MAC determination (one treated and one naive group of halothane and one treated and one naive group of isoflurane). Halothane or isoflurane MAC determination was performed before (basal) and 30 min after an intraperitoneal dose of 30 μg/kg of dexmedetomidine (post-dex) from alveolar gas samples at the time of tail clamp. Administration of an acute dose of dexmedetomidine to animals that had chronically received dexmedetomidine resulted in a MAC-sparing effect that was similar to that seen in naive animals for halothane; however, the same treatment increased the MAC-sparing response of dexmedetomidine for isoflurane. Isoflurane but not halothane MAC-sparing response of acutely administered dexmedetomidine is enhanced in rats chronically treated with this drug.     

Neuroprotection by methanol extract of Uncaria rhynchophylla against global cerebral ischemia in rats

In traditional Oriental medicine, Uncaria rhynchophylla has been used to lower blood pressure and to relieve various neurological symptoms. However, scientific evidence related to its effectiveness or precise modes of action has not been available. Thus, in the current study, we evaluated neuroprotective effects of U. rhynchophylla after transient global ischemia using 4-vessel occlusion model in rats. Methanol extract of U. rhynchophylla administered intraperitoneally (100-1000 mg/kg at 0 and 90 min after reperfusion) significantly protected hippocampal CA1 neurons against 10 min transient forebrain ischemia. Measurement of neuronal cell density in CA1 region at 7 days after ischemia by Nissl staining revealed more than 70% protection in U. rhynchophylla-treated rats compared to saline-treated animals. In U. rhynchophylla-treated animals, induction of cyclooxygenase-2 in hippocampus at 24 hr after ischemia was significantly inhibited at both mRNA and protein levels. Furthermore, U. rhynchophylla extract inhibited TNF-alpha and nitric oxide production in BV-2 mouse microglial cells in vitro. These anti-inflammatory actions of U. rhynchophylla extract may contribute to its neuroprotective effects.     

Suppression of lordosis in guinea pigs by ethamoxy-triphetol (MER-25) given at long intervals (34-46 hr) after estradiol benzoate treatment

Ovariectomized guinea pigs were given estradiol benzoate (EB) followed 40 hr later by progesterone (P). Behavioral testing commenced 1 hr after P injection and continued at hourly intervals for 8 hr. This treatment activated lordosis in almost 100% of animals. Administration of the antiestrogen MER-25 (75 mg/kg body wt per injection) between 2 hr before and 6 hr after EB treatment did not cause a significant decline in proportion of animals displaying lordosis, but did cause a decrease in length of time the lordosis position was held (maximum lordosis, sec). In contrast, $\text{13}\text{14}$ animals given MER-25 at 2 hr before and 2 hr after P and $\text{8}\text{10}$ animals given MER-25 simultaneously with and 2 hr after P, failed to show lordosis. Administration of supplementary EB at around the time of P injection, partially alleviated these behavior-blocking effects of MER-25. When MER-25 was given 2–6 hr after administration of P there was a significant decrease in duration of heat (hr). These results suggest that in addition to its early “triggering” effects, estrogen has important “maintenance” effects which determine the character of heat in guinea pigs. Continued presence of estrogen in the nervous system may be a requirement for the facilitatory actions of P on sexual behavior in guinea pigs, but such a requirement may not exist in other rodents such as rats.     

Aminoguanidine Administration Ameliorates Hippocampal Damage After Middle Cerebral Artery Occlusion in Rat

The effects of a selective inducible nitric oxide synthase inhibitor aminoguanidine (AG) on neuronal cells survival in hippocampal CA1 region after middle cerebral artery occlusion (MCAO) were examined. Transient focal cerebral ischemia was induced in rats by 60 or 90 min of MCAO, followed by 7 days of reperfusion. AG treatment (150 mg/kg i.p.) significantly reduced total infarct volumes: by 70% after 90 min MCAO and by 95% after 60 min MCAO, compared with saline-treated ischemic group. The number of degenerating neurons in hippocampal CA1 region was also markedly lower in aminoguanidine-treated ischemic groups compared to ischemic groups without AG-treatment. The number of iNOS-positive cells significantly increased in the hippocampal CA1 region of ischemic animals, whereas it was reduced in AG-treated rats. Our findings demonstrate that aminoguanidine decreases ischemic brain damage and improves neurological recovery after transient focal ischemia induced by MCAO.     

Effects of L-arginine on prevention and treatment of lithium-pilocarpine-induced status epilepticus

The effects of various doses of L-arginine, a nitric oxide substrate, on lithium-pilocarpine-induced seizures were studied in rats. Rats were implanted with chronic, stainless steel screw electrodes epidurally for electrocortical recordings. A control group received 3 mEq/kg LiCl (i.p.) and 24 h later 45 mg/kg pilocarpine HCl (i.p.). Two different experimental procedures were followed: (1) L-arginine was applied in doses of 100 mg/kg, 300 mg/kg or 500 mg/kg (i.p.), 30 min before pilocarpine injection; (2) 300 mg/kg, 500 mg/kg or 1000 mg/kg (i.p.) L-arginine was injected either 5 min or 30 min after the onset of status epilepticus (SE). L-arginine (300 mg/kg) injected 30 min before pilocarpine significantly reduced the percentage of SE, but did not change the latency to SE or 24-hour survival. These parameters were not significantly affected by the 100 mg/kg or 500 mg/kg dose of L-arginine. On the other hand, no dose of L-arginine that was applied after SE had begun, had any significant influence on the seizures. We concluded that L-arginine may prevent seizure activity in some but not all doses, and does not have any effect on the ongoing seizure activity.     

Loss of Hippocampal Neurons after Kainate Treatment Correlates with Behavioral Deficits

Treating rats with kainic acid induces status epilepticus (SE) and leads to the development of behavioral deficits and spontaneous recurrent seizures later in life. However, in a subset of rats, kainic acid treatment does not induce overt behaviorally obvious acute SE. The goal of this study was to compare the neuroanatomical and behavioral changes induced by kainate in rats that developed convulsive SE to those who did not. Adult male Wistar rats were treated with kainic acid and tested behaviorally 5 months later. Rats that had experienced convulsive SE showed impaired performance on the spatial water maze and passive avoidance tasks, and on the context and tone retention tests following fear conditioning. In addition, they exhibited less anxiety-like behaviors than controls on the open-field and elevated plus-maze tests. Histologically, convulsive SE was associated with marked neuron loss in the hippocampal CA3 and CA1 fields, and in the dentate hilus. Rats that had not experienced convulsive SE after kainate treatment showed less severe, but significant impairments on the spatial water maze and passive avoidance tasks. These rats had fewer neurons than control rats in the dentate hilus, but not in the hippocampal CA3 and CA1 fields. Correlational analyses revealed significant relationships between spatial memory indices of rats and neuronal numbers in the dentate hilus and CA3 pyramidal field. These results show that a part of the animals that do not display intense behavioral seizures (convulsive SE) immediately after an epileptogenic treatment, later in life, they may still have noticeable structural and functional changes in the brain.     

Silencing of P2X7R by RNA interference in the hippocampus can attenuate morphological and behavioral impact of pilocarpine-induced epilepsy

Cell signaling mediated by P2X7 receptors (P2X7R) has been suggested to be involved in epileptogenesis, via modulation of intracellular calcium levels, excitotoxicity, activation of inflammatory cascades, and cell death, among other mechanisms. These processes have been described to be involved in pilocarpine-induced status epilepticus (SE) and contribute to hyperexcitability, resulting in spontaneous and recurrent seizures. Here, we aimed to investigate the role of P2X7R in epileptogenesis in vivo using RNA interference (RNAi) to inhibit the expression of this receptor. Small interfering RNA (siRNA) targeting P2X7R mRNA was injected into the lateral ventricles (icv) 6 h after SE. Four groups were studied: Saline-Vehicle, Saline-siRNA, Pilo-Vehicle, and Pilo-siRNA. P2X7R was quantified by western blotting and neuronal death assessed by Fluoro-Jade B histochemistry. The hippocampal volume (edema) was determined 48 h following RNAi. Behavioral parameters as latency to the appearance of spontaneous seizures and the number of seizures were determined until 60 days after the SE onset. The Saline-siRNA and Pilo-siRNA groups showed a 43 and 37% reduction, respectively, in P2X7R protein levels compared to respective vehicle groups. Neuroprotection was observed in CA1 and CA3 of the Pilo-siRNA group compared to Pilo-Vehicle. P2X7R silencing in pilocarpine group reversed the increase in the edema detected in the hilus, suprapyramidal dentate gyrus, CA1, and CA3; reduced mortality rate following SE; increased the time to onset of spontaneous seizure; and reduced the number of seizures, when compared to the Pilo-Vehicle group. Therefore, our data highlights the potential of P2X7R as a therapeutic target for the adjunct treatment of epilepsy.     

Effect of chronic underfeeding on uterine glycogen of rats. Influence of indomethacin and nordihydroguaiaretic acid

Glycogen values in uterine strips isolated from normal-fed estrous or diestrous rats, or from rats fed a restricted diet (50% of normal food intake for 25 days) were measured. Determinations were made immediately after killing (0 time or post-isolation) as well as after incubation in glucose-free medium (60 min time or post-incubation). The post-incubation levels of glycogen in the uteri from normal-fed animals diminished significantly in comparison to post-isolation values, and this decrement was not modified by the addition of indomethacin, nordihydroguaiaretic acid or exogenous prostaglandins E1, E2 or F2 alpha. In rats fed a restricted-diet, the initial glycogen values (0 time) were significantly lower than in normal-fed controls, but did not decline further after incubation in glucose-free medium (60 min time). The addition of indomethacin, acetylsalicylic acid or of nordihydroguaiaretic acid led to a significant fall in the glycogen levels, and exogenous PGE1, PGE2 or PGF2 alpha failed to alter the effects of the inhibitors. The values of PGE and PGF prostaglandins release to the medium by the uterus from restricted-diet rats did not differ from those obtained in the experiments with normal-fed animals. Administration of 17-beta estradiol to restricted-diet rats led to suppression of the effects of this diet on the glycogen concentration. The above results indicate that in rats subjected to a prolonged period of dietary restriction, the uterine glycogen becomes responsive to the effects of cyclooxygenase and lipoxygenase inhibitors, suggesting the operation of some regulatory mechanism during critical periods of nutrition.     

Caerulein and secretin induced pancreatic growth: a possible control by endogenous pancreatic somatostatin

Pancreatic hypertrophy and hyperplasia following chronic joint (CA + SE), or separate, caerulein (CA: 1 microgram . kg-1) and secretin (SE: 75 micrograms . kg-1) administration were studied in parallel with pancreatic somatostatin (SRIF) contents following 2, 4, 7 and 10 days of treatment. Parameters indicative of pancreatic growth (tissue weight, DNA and protein contents, cellular protein concentrations) increased significantly after 2 days of CA or CA + SE and reached a plateau between days 4 and 10. SE merely induced a mild hypertrophy after 4 days. Endogenous pancreatic SRIF contents varied upon treatment, differently so with each peptide regimen. Indeed, CA and CA + SE treatments decreased total SRIF contents after 2 days with no effect thereafter. SE also decreased the latter after 2 days while significant increases were observed after 7 and 10 days. The inverse relationship seemingly existing between SRIF contents and the amplitude of hormonally-induced pancreatic growth supports the hypothesis that endogenous pancreatic SRIF, operating as an 'antigrowth' factor, may participate in the exogenous CA, SE and CA + SE stimulated pancreatic growth phenomena.     

Effects of α-Phenyl-N-tert-butyl Nitrone (PBN) on Compression Injury of Rat Spinal Cord

-Phenyl-N-tert-butyl Nitrone (PBN) is a free radical scavenger which recently has proved to be neuroprotective in experimental studies on focal cerebral ischemia and infarction. We therefore studied the effect of this drug in a model of moderate compression injury to rat spinal cord at the midthoracic level. The compound was given intraperitoneally 0.5 h before (100 mg/kg b.w) and at 1.5 h (50 mg/kg b.w) and 3.5 h (50 mg/kg b.w) after compression. Treated animals and controls (vehicle alone) were allowed to survive for 1 or 9 days following trauma. The functional outcome was tested by the inclined plane method and the motor performance score. By using MAP2 immuno-staining the number of nerve cell bodies in the ventral horn and the ratio of MAP2 immunostained area to area of whole section of the cord were assessed to detect loss of neurons and loss of dendrites in the compressed segment. pAPP and PGP9.5 immunostaining was used to demonstrate axonal lesions.

Treated and control rats showed at day 1 when tested with the inclined plane method a marked reduction of the capacity angle. This abnormality recovered gradually over the following days and was normalized at day 9. The motor performance score showed a marked reduction at day 1 which almost normalized at day 9. There was no difference regarding the functional outcome between rats given PBN and controls in none one of these functional tests.

The spinal cord of normal rats presented immunoreactivity to MAP2 in nerve cell bodies and dendrites but not in axons and other structures. Following compression there was at day 1 and 9 a marked loss of MAP2 immunoreactivity in dendrites and nerve cell bodies. We could not detect any difference between the PBN and the control rats regarding the degree of cell loss or degree of reduction of dendrite staining. No difference between the two groups was seen with the axonal immunostainings (βAPP and PGP9.5).

In conclusion, our study did not reveal any neuroprotective effect of PBN on the functional outcome and morphology (immunostaining to MAP2, pAPP and PGP9.5) in this model of moderate compression trauma to rat spinal cord.