Plasma concentrations of buprenorphine following a single subcutaneous administration of a sustained release formulation of buprenorphine in sheep.

The goal of the present study was to evaluate the potential use of slow release buprenorphine in sheep. Twelve adult female sheep (6 Dorset and 6 Suffolk, 12 months of age) were used for this project and were divided into 2 experimental groups (n = 6/group comprising 3 Dorset and 3 Suffolk sheep). Sustained release (SR) buprenorphine was administered subcutaneously in the scapular region at a concentration of 0.1 mg/kg body weight (BW) for group 1 and of 0.05 mg/kg BW for group 2. Following blood collections at selected time points, plasma concentrations of buprenorphine was performed by tandem liquid chromatograph-mass spectrometry. Mean buprenorphine concentration was above 0.1 ng/mL at 48 h up to 192 h post-injection for group 1 and it was above 0.1 ng/mL at 48 h up to 72 h post-injection for group 2. In conclusion, a long lasting potential analgesic plasma level of buprenorphine is attained following a single subcutaneous injection of 0.1 mg/kg BW of SR buprenorphine in sheep. However the effective analgesic plasma threshold still needs to be determined in sheep.

L'objectif de la présente étude était d'évaluer l'utilisation potentielle de buprénorphine à relâchement lent (RL) chez le mouton. Douze brebis adultes (6 Dorset et 6 Suffolk, 12 mois d'âge) ont été utilisées pour ce projet et ont été réparties en deux groupes expérimentaux (n = 6/groupe, 3 Dorset et 3 Suffolk). De la buprénorphine à relâchement continu a été administrée par voie sous-cutanée dans la région scapulaire à une concentration de 0,1 mg/kg de poids corporel (PC) pour le groupe 1 et à 0,05 mg/kg de PC pour le groupe 2. Suite à des prélèvements sanguins à des moments sélectionnés, les concentrations plasmatiques de buprénorphine ont été déterminées par spectrométrie de masse en tandem avec la chromatographie en phase liquide. La concentration moyenne de buprénorphine était supérieure à 0,1 ng/mL après 48 h et jusqu'à 192 h post-injection pour le groupe 1, et était supérieure à 0,1 ng/mL après 48 et jusqu'à 72 h post-injection pour le groupe 2. En conclusion, un niveau plasmatique prolongé de buprénorphine avec un potentiel analgésique est atteint suite à une injection sous-cutanée unique de 0,1 mg/kg de PC de buprénorphine RL chez le mouton. Toutefois, le seuil plasmatique analgésique réel demeure encore à être déterminé chez le mouton.(Traduit par Docteur Serge Messier).

Early-life stress is associated with gender-based vulnerability to epileptogenesis in rat pups.

During development, the risk of developing mesial temporal lobe epilepsy (MTLE) increases when the developing brain is exposed to more than one insult in early life. Early life insults include abnormalities of cortical development, hypoxic-ischemic injury and prolonged febrile seizures. To study epileptogenesis, we have developed a two-hit model of MTLE characterized by two early-life insults: a freeze lesion-induced cortical malformation at post-natal day 1 (P1), and a prolonged hyperthermic seizure (HS) at P10. As early life stressors lead to sexual dimorphism in both acute response and long-term outcome, we hypothesized that our model could lead to gender-based differences in acute stress response and long-term risk of developing MTLE. Male and female pups underwent a freeze-lesion induced cortical microgyrus at P1 and were exposed to HS at P10. Animals were monitored by video-EEG from P90 to P120. Pre and post-procedure plasma corticosterone levels were used to measure stress response at P1 and P10. To confirm the role of sex steroids, androgenized female pups received daily testosterone injections to the mother pre-natally and post-natally for nine days while undergoing both insults. We demonstrated that after both insults females did not develop MTLE while all males did. This correlated with a rise in corticosterone levels at P1 following the lesion in males only. Interestingly, all androgenized females showed a similar rise in corticosterone at P1, and also developed MTLE. Moreover, we found that the cortical lesion significantly decreased the latency to generalized convulsion during hyperthermia at P10 in both genders. The cortical dysplasia volumes at adulthood were also similar between male and female individuals. Our data demonstrate sexual dimorphism in long-term vulnerability to develop epilepsy in the lesion + hyperthermia animal model of MTLE and suggest that the response to early-life stress at P1 contributes significantly to epileptogenesis in a gender-specific manner.

Comparative vascular responses three months after paclitaxel and everolimus-eluting stent implantation in streptozotocin-induced diabetic porcine coronary arteries.

BACKGROUND: Diabetes remains a significant risk factor for restenosis/thrombosis following stenting. Although vascular healing responses following drug-eluting stent (DES) treatment have been characterized previously in healthy animals, comparative assessments of different DES in a large animal model with isolated features of diabetes remains limited. We aimed to comparatively assess the vascular response to paclitaxel-eluting (PES) and everolimus-eluting (EES) stents in a porcine coronary model of streptozotocin (STZ)-induced type I diabetes. METHOD: Twelve Yucatan swine were induced hyperglycemic with a single STZ dose intravenously to ablate pancreatic ß-cells. After two months, each animal received one XIENCE V® (EES) and one Taxus Liberte (PES) stent, respectively, in each coronary artery. After three months, vascular healing was assessed by angiography and histomorphometry. Comparative in vitro effects of everolimus and paclitaxel (10-5 M-10-12 M) after 24 hours on carotid endothelial (EC) and smooth muscle (SMC) cell viability under hyperglycemic (42 mM) conditions were assayed by ELISA. Caspase-3 fluorescent assay was used to quantify caspase-3 activity of EC treated with everolimus or paclitaxel (10-5 M, 10-7 M) for 24 hours. RESULTS: After 3 months, EES reduced neointimal area (1.60 ± 0.41 mm, p < 0.001) with trends toward reduced % diameter stenosis (11.2 ± 9.8%, p = 0.12) and angiographic late-loss (0.28 ± 0.30 mm, p = 0.058) compared to PES (neointimal area: 2.74 ± 0.58 mm, % diameter stenosis: 19.3 ± 14.7%, late loss: 0.55 ± 0.53 mm). Histopathology revealed increased inflammation scores (0.54 ± 0.21 vs. 0.08 ± 0.05), greater medial necrosis grade (0.52 ± 0.26 vs. 0.0 ± 0.0), and persistently elevated fibrin scores (1.60 ± 0.60 vs. 0.63 ± 0.41) with PES compared to EES (p < 0.05). In vitro, paclitaxel significantly increased (p < 0.05) EC/SMC apoptosis/necrosis at high concentrations (≥ 10-7 M), while everolimus did not affect EC/SMC apoptosis/necrosis within the dose range tested. In ECs, paclitaxel (10-5 M) significantly increased caspase-3 activity (p < 0.05) while everolimus had no effect. CONCLUSION: After 3 months, both DES exhibited signs of delayed healing in a STZ-induced diabetic swine model. PES exhibited greater neointimal area, increased inflammation, greater medial necrosis, and persistent fibrin compared to EES. Differential effects of everolimus and paclitaxel on vascular cell viability may potentially be a factor in regulating delayed healing observed with PES. Further investigation of molecular mechanisms may aid future development of stent-based therapies in treating coronary artery disease in diabetic patients.

Mice doubly-deficient in lysosomal hexosaminidase A and neuraminidase 4 show epileptic crises and rapid neuronal loss.

Tay-Sachs disease is a severe lysosomal disorder caused by mutations in the HexA gene coding for the α-subunit of lysosomal ß-hexosaminidase A, which converts G(M2) to G(M3) ganglioside. Hexa(-/-) mice, depleted of ß-hexosaminidase A, remain asymptomatic to 1 year of age, because they catabolise G(M2) ganglioside via a lysosomal sialidase into glycolipid G(A2), which is further processed by ß-hexosaminidase B to lactosyl-ceramide, thereby bypassing the ß-hexosaminidase A defect. Since this bypass is not effective in humans, infantile Tay-Sachs disease is fatal in the first years of life. Previously, we identified a novel ganglioside metabolizing sialidase, Neu4, abundantly expressed in mouse brain neurons. Now we demonstrate that mice with targeted disruption of both Neu4 and Hexa genes (Neu4(-/-);Hexa(-/-)) show epileptic seizures with 40% penetrance correlating with polyspike discharges on the cortical electrodes of the electroencephalogram. Single knockout Hexa(-/-) or Neu4(-/-) siblings do not show such symptoms. Further, double-knockout but not single-knockout mice have multiple degenerating neurons in the cortex and hippocampus and multiple layers of cortical neurons accumulating G(M2) ganglioside. Together, our data suggest that the Neu4 block exacerbates the disease in Hexa(-/-) mice, indicating that Neu4 is a modifier gene in the mouse model of Tay-Sachs disease, reducing the disease severity through the metabolic bypass. However, while disease severity in the double mutant is increased, it is not profound suggesting that Neu4 is not the only sialidase contributing to the metabolic bypass in Hexa(-/-) mice.

N-methyl-D-aspartate, hyperpolarization-activated cation current (Ih) and gamma-aminobutyric acid conductances govern the risk of epileptogenesis following febrile seizures in rat hippocampus.

Febrile seizures are the most common types of seizure in children, and are generally considered to be benign. However, febrile seizures in children with dysgenesis have been associated with the development of temporal lobe epilepsy. We have previously shown in a rat model of dysgenesis (cortical freeze lesion) and hyperthermia-induced seizures that 86% of these animals developed recurrent seizures in adulthood. The cellular changes underlying the increased risk of epileptogenesis in this model are not known. Using whole cell patch-clamp recordings from CA1 hippocampal pyramidal cells, we found a more pronounced increase in excitability in rats with both hyperthermic seizures and dysgenesis than in rats with hyperthermic seizures alone or dysgenesis alone. The change was found to be secondary to an increase in N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs). Inversely, hyperpolarization-activated cation current was more pronounced in naïve rats with hyperthermic seizures than in rats with dysgenesis and hyperthermic seizures or with dysgenesis alone. The increase in GABAA-mediated inhibition observed was comparable in rats with or without dysgenesis after hyperthermic seizures, whereas no changes were observed in rats with dysgenesis alone. Our work indicates that in this two-hit model, changes in NMDA receptor-mediated EPSCs may facilitate epileptogenesis following febrile seizures. Changes in the hyperpolarization-activated cation currents may represent a protective reaction and act by damping the NMDA receptor-mediated hyperexcitability, rather than converting inhibition into excitation. These findings provide a new hypothesis of cellular changes following hyperthermic seizures in predisposed individuals, and may help in the design of therapeutic strategies to prevent epileptogenesis following prolonged febrile seizures.

Pharmacokinetics of vanillin and its effects on mechanical hypersensitivity in a rat model of neuropathic pain.

The analgesic effects of vanillin on neuropathic pain was evaluated using thermal sensitivity and mechanical allodynia using the sciatic nerve constriction model (n = 30 rats). To determine the pharmacokinetics of vanillin, rats (n = 6/administration route) received either 20 or 100 mg/kg of vanillin i.v. and p.o., respectively. For the pharmacodynamic study, baseline levels for hyperalgesia and allodynia were taken for 5 days prior to surgery. Following surgery each group (n = 6 rats/group) received either vanillin (50 mg/kg or 100 mg/kg), morphine (2 mg/kg or 6 mg/kg) or the vehicle only. Pharmacokinetic results following p.o. administrations are C(max) 290.24 ng/mL, T(max) 4 h, relative clearance 62.17 L/h/kg and T(1/2) 10.3 h. The bioavailability is 7.6%. Mechanical allodynia was decreased on treatment days 1, 2, 3, 5 (p < 0.003) and not on day 4 (p > 0.02) with 50 mg/kg vanillin, whereas at 100 mg/kg p.o. a decrease was noted only on days 7 and 8 (p < 0.003). No effect on hyperalgesia was seen following vanillin administration. In conclusion, vanillin is bioavailable and seems to have an alleviating effect on mechanical allodynia, and not on hyperalgesia, when evaluated with a chronic constriction nerve injury rat model of neuropathic pain.

Effects of timing of dexamethasone treatment on the outcome of collagenase-induced intracerebral hematoma in rats.

The effect of timing in providing dexamethasone treatment after intracerebral hematoma was evaluated in rats with hematoma induced by a subcortical collagenase injection. Male Sprague-Dawley rats (n = 30; body weight, 185 to 230 g) received dexamethasone (1 mg/kg) intraperitoneally at 2 h, 4 h, or 6 h (1 group per time point) after intracerebral collagenase injection, with another dose (1 mg/kg) administered at 24 h after collagenase injection. Neurologic examinations and rotarod treadmill tests were used to evaluate motor behavior before and at 24 and 48 h after intracerebral injection. Rats were euthanized after the last behavioral test. Brains were evaluated for hematoma size, number of penumbral necrotic neurons, neutrophils within the hematoma, and astrocytic response. Compared with the control and other treatment groups, rats treated with dexamethasone at 2 and 24 h after intracerebral collagenase injection scored significantly better on neurologic exams and rotarod tests. Hematoma volume was significantly smaller in all treated groups than in the control group but did not differ between treatment groups. Fewer neutrophils were seen in the perihematoma region of all treated rats compared with controls, but the number of necrotic neurons was decreased significantly only in the group treated with dexamethasone at 2 and 24 h. These results indicate that a 1-mg/kg dose of dexamethasone is beneficial for treatment of intracerebral hemorrhage, particularly if administered early after the hemorrhagic insult.

Synchronized gamma oscillations (30-50 Hz) in the amygdalo-hippocampal network in relation with seizure propagation and severity.

In this study, we demonstrate that gamma oscillations (30-50 Hz) recorded in the local field potentials (LFP) of the hippocampus are a marker of temporal lobe seizure propagation and that the level of LFP synchrony in the amygdalo-hippocampal network, during these oscillations, is related to the severity of seizures. Sprague-Dawley rats were given a single systemic dose of kainic acid (KA; 6 mg/kg, i.p.) and local field potential activity (1-475 Hz) of the dorsal hippocampus, the amygdala and the neocortex was recorded. Of 135 ictal discharges, 55 (40.7%) involved both limbic structures. We demonstrated that 78.2% of seizures involving both the hippocampus and amygdala showed hippocampal gamma oscillations. Seizure duration was also significantly correlated with the frequency of hippocampal gamma oscillations (r2=0.31, p<0.01) and LFP synchrony in the amygdalo-hippocampal network (r2=0.21, p<0.05). These results suggest that gamma oscillations in the amygdalo-hippocampal network could facilitate long-range synchrony and participate in the propagation of seizures.

Hippocampal atrophy and abnormal brain development following a prolonged hyperthermic seizure in the immature rat with a focal neocortical lesion.

In rats subjected to a focal cortical lesion soon after birth, hyperthermia at P10 induces a prolonged epileptic seizure, often followed by temporal lobe epilepsy in the adult. To determine whether brain damage and notably hippocampal atrophy occur early on in this model, whole brain as well as hemispheric, cortical, subcortical and hippocampal volumes was measured in non-lesioned and lesioned rat pups, 2 days (P12) and 12 days (P22) after the hyperthermic seizure. All pups with a cortical lesion showed reductions in whole brain and in ipsilateral hemispheric, cortical and hippocampal volumes at P12, which persisted at P22 in pups having also sustained a prolonged hyperthermic seizure at P10. Limiting the duration of the seizure with Diazepam prevented the hippocampal atrophy. Thus, a prolonged hyperthermic seizure in immature brain with a subtle neocortical lesion impairs normal brain development, and the duration of the seizure appears to be a key factor in generating hippocampal atrophy.

Febrile seizures in the predisposed brain: a new model of temporal lobe epilepsy.

The atypical febrile seizure has important clinical implications because of its association with the mesial temporal lobe epilepsy syndrome, which is the most common of the intractable epilepsies. However, whether a causal relation exists between these conditions is currently unknown. We have previously shown that a focal cortical lesion induced in the neonatal rat predisposes to the development of atypical hyperthermic seizures. We show here that 86% of the lesion plus hyperthermia group experience development of spontaneous recurrent seizures recorded from the amygdala ipsilateral to the lesion. Control rats did not have spontaneous recurrent behavioral or electrographic seizures. Lesioned rats with hyperthermic seizures also showed an impaired performance on the Morris water maze when compared with naive control rats, suggesting mild deficits in learning and memory. These findings support a link between the atypical febrile seizure and mesial temporal lobe epilepsy, and at the same time establish a new model for this condition through which new preventative and therapeutic strategies can be tested.

High doses of methylprednisolone are required for the treatment of collagenase-induced intracerebral hemorrhage in rats.

Methylprednisolone (MP) was evaluated for the treatment of intracerebral hemorrhage in a Sprague-Dawley rat model of cerebral hematoma induced by subcortical injection of collagenase. At 1 and 24 h after the injection, MP was administered intraperitoneally (IP) at a concentration of 10, 35, or 100 mg/kg. Control groups received saline IP at 1 and 24 h after the intracerebral injection of collagenase (positive controls) or saline (negative controls). Motor behaviour 24 h before and 24 h and 48 h after the intracerebral injection was evaluated by means of a neurologic exam and a rotarod treadmill test. The animals were euthanized at 48 h; brain water content was determined in half of the rats, and histopathological studies were done in the other half. Compared with the positive controls, the animals with collagenase-induced hematoma performed significantly better on the neurologic exam after treatment with 100 mg/kg of MP and on the rotarod test after treatment with 35 or 100 mg/kg of MP. The hematoma volume was significantly smaller (P < 0.002) after all doses of MP; however, the smallest volume was seen with 100 mg/kg. There were significantly fewer neutrophils (P < 0.01) within the hematoma in the MP-treated animals (maximum reduction with 100 mg/kg) than in the positive controls, but the numbers of reactive astrocytes did not differ significantly between the treatment groups. The number of necrotic neurons in the penumbra did not differ between the treatment groups; however, there were significantly fewer (P < 0.005) in the cerebral cortex in the group treated with 100 mg/kg of MP compared with the positive controls. These results suggest that high doses of MP administered shortly after occurrence of a cerebral hematoma are beneficial for the treatment of intracerebral hemorrhage.