conotoxin-gv and Infarction--Middle-Cerebral-Artery

The potent NMDA receptor antagonist, Conantokin-G (CGX-1007), a snail peptide, has an 8-h therapeutic window in rat focal cerebral ischemia. We hypothesized that the mechanism of neuroprotection is the inhibition of 'secondary phase' peri-infarct depolarizations (PIDs), recently shown to recur 6--24 h post-reperfusion. Rats were implanted with intrathecal (i.t.) catheters for drug delivery and DC-compatible electrodes for continuous PID monitoring and subjected to transient (2 h) middle cerebral artery occlusion. Four groups were studied. In two groups (C(40)C and C(20)C), continuous infusion of CGX--1007 was administered over 8--24 h post-occlusion at 0.1 microg/h (0.04 nmol/h) following either a 40- or 20-nmol bolus dose at 8 h. Another group (C(40)S) received the 40-nmol bolus followed by saline infusion, and a control group received saline. Intrathecal drug treatment reduced infarct volumes relative to controls by 61%, 31%, and 10% in C(40)C, C(40)S, and C(20)C groups, respectively, but also induced dose-dependent paralysis and elevated mortality. All rats had PID rates similar to the control group prior to treatment, but following treatment secondary phase PIDs were reduced by 47--57% in each drug group compared to controls. Because several animals exhibited PID inhibition but no neuroprotection, there was no significant correlation between these endpoints across groups. However, drug-treated animals that did not exhibit secondary phase PIDs prior to treatment had significantly smaller infarcts and reduced subsequent PID activity than corresponding control rats. Results suggest that post-reperfusion PIDs play a substantial, though still undefined pathogenic role in delayed maturation of cerebral infarction and NMDA receptor-targeted neuroprotection.

Topics: Analysis of Variance; Animals; Body Temperature; Body Weight; Brain Infarction; Conotoxins; Cortical Spreading Depression; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Electrodes; Excitatory Amino Acid Antagonists; Infarction, Middle Cerebral Artery; Ischemia; Male; Rats; Rats, Sprague-Dawley; Reperfusion; Time Factors

Delayed cell death following ischemic brain injury has been linked to alterations in gene expression. In this study we have evaluated the upregulation of several genes associated with delayed cell death (c-fos, bax, and bcl-2) during the initial 24 h of transient middle cerebral artery occlusion (MCAo) in the rat and the effects of postinjury treatment with the NR2B subunit specific NMDA receptor antagonist CGX-1007 (Conantokin-G, Con-G). C-fos mRNA levels peaked at 1 h postinjury in both cortical and subcortical ischemic brain regions (30-fold increase), remained elevated at 4 h and returned to within normal, preinjury levels 24 h postinjury. The increase in mRNA levels correlated to increased protein expression in the entire ipsilateral hemisphere at 1 h. Regions of necrosis at 4 h were void of C-Fos immunoreactivity with continued upregulation in surrounding regions. At 24 h, loss of C-Fos staining was observed in the injured hemisphere except for sustained increases along the border of the infarct and in the cingulate cortex of vehicle treated rats. CGX-1007 treatment reduced c-fos expression throughout the infarct region by up to 50%. No significant differences were measured in either bcl-2 or bax mRNA expression between treatment groups. However, at 24 h postinjury CGX-1007 treatment was associated with an increase in Bcl-2 immunoreactivity that correlated to a reduction in DNA fragmentation. In conclusion, CGX-1007 effectively attenuated gene expression associated with delayed cell death as related to a neuroprotective relief of cerebral ischemia.

Topics: Animals; Antibodies; bcl-2-Associated X Protein; Brain Ischemia; Cell Death; Conotoxins; DNA Damage; Excitatory Amino Acid Antagonists; Gene Expression; Histocytochemistry; Infarction, Middle Cerebral Artery; Male; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; RNA, Messenger

In focal cerebral ischemia, peri-infarct depolarizations (PIDs) cause an expansion of core-infarcted tissue into adjacent penumbral regions of reversible injury and have been shown to occur through 6 hr after injury. However, infarct maturation proceeds through 24 hr. Therefore, we studied PID occurrence through 72 hr after both transient and permanent middle cerebral artery occlusion (MCAo) via continuous DC recordings in nonanesthetized rats. PIDs occurred an average 13 times before reperfusion at 2 hr and then ceased for an average approximately 8 hr. After this quiescent period, PID activity re-emerged in a secondary phase, which reached peak incidence at 13 hr and consisted of a mean 52 PIDs over 2-24 hr. This phase corresponded to the period of infarct maturation; rates of infarct growth through 24 hr coincided with changes in PID frequency and peaked at 13 hr. In permanent MCAo, PIDs also occurred in a biphasic pattern with a mean of 78 events over 2-24 hr. Parameters of secondary phase PID incidence correlated with infarct volumes in transient and permanent ischemia models. The role of secondary phase PIDs in infarct development was further investigated in transient MCAo by treating rats with a high-affinity NMDA receptor antagonist at 8 hr after injury, which reduced post-treatment PID incidence by 57% and provided 37% neuroprotection. Topographic mapping with multielectrode recordings revealed multiple sources of PID initiation and patterns of propagation. These results suggest that PIDs contribute to the recruitment of penumbral tissue into the infarct core even after the restoration of blood flow and throughout the period of infarct maturation.

Topics: Animals; Brain Ischemia; Cell Death; Conotoxins; Cortical Spreading Depression; Electric Conductivity; Excitatory Amino Acid Antagonists; Infarction, Middle Cerebral Artery; Kinetics; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate

The NMDA antagonist CGX-1007 (Conantokin-G) has previously been shown to possess potent neuroprotective properties when administered intracranially following experimental ischemic brain injury. Using the same model of middle cerebral artery occlusion (MCAo) in rats we now report the neuroprotective effects of CGX-1007 when delivered intrathecally (i.t.). When given 4 h post-occlusion, a reduction in brain infarction was measured along with significant neurological recovery. Furthermore, we describe an i.t. neuroprotective therapeutic window lasting > or = 8 h from the start of the injury. Critically, this is the first comprehensive report of a neuroprotective agent that can be administered i.t. to ameliorate experimental brain injury and potentially provide an excellent therapeutic window as a neuroprotection treatment.

Topics: Animals; Body Weight; Brain Edema; Brain Ischemia; Cerebral Cortex; Conotoxins; Corpus Striatum; Disease Models, Animal; Excitatory Amino Acid Antagonists; Fever; Infarction, Middle Cerebral Artery; Injections, Spinal; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Telencephalon; Treatment Outcome