The Role of Gap Junctions in Brain Glucose Deprivation and Glucose Reperfusion

Hypoglycemia is a severe side effect of insulin overdose in the diabetic population and can result in various neurological sequalae including seizures, coma, and brain death. There is still a limited understanding of the generation and propagation of hypoglycemic seizures, which may exacerbate hypoglycemia-induced neuronal damage. Moreover, glucose reperfusion after a period of transient hypoglycemia has been shown to cause neuronal hyperexcitability which can have further damaging effects. Gap junctional communication can be involved in the spread of hypoglycemic injury in two ways: 1) by providing a cytoplasmic continuity in which seizures can easily propagate and 2) by engaging the astrocytic network in metabolic compensation as well as enhancing astrocytic buffering of K+. This study aims to investigate the role that gap junctions play during brain energy deprivation. Results from these experiments show that gap junction blockade can have a neuroprotective role during hypoglycemia and glucose reperfusion.MAS

Severe Hypoglycemia in a Juvenile Diabetic Rat Model: Presence and Severity of Seizures Are Associated with Mortality

<div><p>It is well accepted that insulin-induced hypoglycemia can result in seizures. However, the effects of the seizures, as well as possible treatment strategies, have yet to be elucidated, particularly in juvenile or insulin-dependent diabetes mellitus (IDDM). Here we establish a model of diabetes in young rats, to examine the consequences of severe hypoglycemia in this age group; particularly seizures and mortality. Diabetes was induced in post-weaned 22-day-old Sprague-Dawley rats by streptozotocin (STZ) administered intraperitoneally (IP). Insulin IP (15 U/kg), in rats fasted (14–16 hours), induced hypoglycemia, defined as <3.5 mM blood glucose (BG), in 68% of diabetic (STZ) and 86% of control rats (CON). Seizures occurred in 86% of STZ and all CON rats that reached hypoglycemic levels with mortality only occurring post-seizure. The fasting BG levels were significantly higher in STZ (12.4±1.3 mM) than in CON rodents (6.3±0.3 mM), resulting in earlier onset of hypoglycemia and seizures in the CON group. However, the BG at seizure onset was statistically similar between STZ (1.8±0.2 mM) and CON animals (1.6±0.1 mM) as well as between those that survived (S+S) and those that died (S+M) post-seizure. Despite this, the S+M group underwent a significantly greater number of seizure events than the S+S group. 25% glucose administered at seizure onset and repeated with recurrent seizures was not sufficient to mitigate these continued convulsions. Combining glucose with diazepam and phenytoin significantly decreased post-treatment seizures, but not mortality. Intracranial electroencephalograms (EEGs) were recorded in 10 CON and 9 STZ animals. Predictive EEG changes were not observed in these animals that underwent seizures. Fluorojade staining revealed damaged cells in non-seizing STZ animals and in STZ and CON animals post-seizure. In summary, this model of hypoglycemia and seizures in juvenile diabetic rats provides a paradigm for further study of underlying mechanisms. Our data demonstrate that severe hypoglycemia (<2.0 mM) is a necessary precondition for seizures, and the increased frequency of these seizures is associated with mortality.</p></div

Effects of treatments (see Table 1) on the seizure scores and mortality in STZ rats.

<p><b>A:</b> Mean number of seizures in (*) <b>glu</b> rats: S+S: 1.6±0.2 (n = 17) and S+M: 4.4±1.2 (n = 5) (p<0.001); <b>ac+1xglu</b> rats: S+S: 1.3±0.2 (n = 8) and S+M: 1.4±0.2 (n = 8). (**) Significant difference also exists between <b>glu</b> rats: S+M and <b>ac+1xglu</b> rats: S+M (p<0.05) <b>B:</b> Mean maximum seizure score attained in <b>glu</b> rats: S+S: 4.3±0.4 (n = 17) and S+M: 5.5±0.4 (n = 5); (*) <b>ac+1xglu</b> rats: S+S: 4.0±0.6 (n = 8) and S+M: 6.1±0.6 (n = 8) (p<0.01).</p

Seizure score to characterize and quantify the observed seizures.

<p>Seizure score to characterize and quantify the observed seizures.</p

Mean blood glucose level measured hourly related to treatment and survival.

<p>S+S Glu: Seizure + Survival; glucose treated.</p><p>S+S AC+1XGLU: Seizure + Survival; glucose and anticonvulsant treated.</p><p>S+M Glu: Seizure + Mortality; glucose treated.</p><p>S+M AC+1XGLU: Seizure + Mortality; glucose and anticonvulsant treated.</p

Comparing the association between seizure severity and mortality in CON and STZ rats.

<p><b>A:</b> No significant difference in the mean score of the first seizure treated in CON rats: S+S: 4.3±0.5 (n = 11) and S+M: 6.0±0.4 (n = 5) and STZ rats: S+S: 3.4±0.3 (n = 17) and S+M: 4.7±0.7 (n = 5) <b>B:</b> No significant difference in the mean maximum seizure score observed in CON rats: S+S: 4.9±0.4 (n = 11) and S+M: 6.5±0.3 and STZ rats: S+S: 4.3±0.4 (n = 17) and S+M: 5.5±0.4 (n = 5) <b>C:</b> A statistically significant difference in the mean number of seizures between (*) CON rats: S+S: 1.6±0.3 (n = 11) and S+M: 7.8±2.7 (n = 5; p<0.01) and between (*) STZ rats: S+S: 1.6±0.2 (n = 17) and S+M: 4.4±1.2 (n = 5; p<0.001).</p

EEG abnormalities during hypoglycemia is not associated with seizure behavior.

<p><b>A:</b> Representative EEG recording of hippocampus (CA1) at baseline (post-fasting; prior to insulin IP), 2 hours, 3 hours (slower waves) and 3.5 (EEG suppression) hours after insulin IP <b>B:</b> Electrographic seizure activity observed in CA1 (lower trace) after suppression of EEG activity. No ictal activity in cortex (upper trace) <b>C:</b> EEG recording of hippocampus and contralateral MRF obtained in STZ rat during hypoglycemia. Electrographic seizure activity observed after suppression of EEG activity. Lower trace illustrates magnification of the area in the gray box <b>D:</b> EEG of the rat in (<b>C</b>) during a behavioural seizure; ictal activity may be masked by movement artifact.</p

Mean blood glucose level measured hourly related to outcome; (±S.E.M).

<p>NS: No Seizure.</p><p>S+M: Seizure + Mortality.</p><p>S+S: Seizure + Survival.</p

Effects of diabetes and seizures on neuronal damage.

<p><b>A:</b> Significant difference in the number of fluorojade (+) cells between CON non-seizing (C+NS: 0±0; n = 5 animals) and STZ non-seizing groups (D+NS; 2.17±1.9 cells; n = 6; p<0.05). No significant difference in the number of cells between CON seizing (C+S: 1.4±1.5; n = 5 cells) and STZ seizing groups (D+S: 4.7±5.8 cells; n = 7) <b>B:</b> Fluorojade (+) cells in the cortical region of (D+S) rat magnified 40X (white arrows).</p

Efficacy of treatment strategies (see Table 1) in preventing subsequent seizures and mortality in STZ rats.

<p><b>A</b>: BG decrease is not significantly different in seizing animals regardless of treatment; <b>glu</b>: seizure+ survival (S+S Glu), <b>ac+1xglu</b>: S+S AC, <b>glu</b>: seizure + mortality (S+M Glu), <b>ac+1xglu</b>: S+M AC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083168#pone-0083168-t004" target="_blank">Table 4</a>). B: No significant difference in the incidence of seizures post-treatment in <b>ac+1xglu</b>: 24% (n = 5/21) compared with glu: 55% (n = 12/22) (p<0.05). (*) Significantly lower incidence of seizures post-treatment in <b>ac+multiple glu</b>: 15% (n = 2/14) compared with <b>glu</b>: 55% (n = 12/22) (p<0.02) C: (*) Significantly higher survival rate in <b>ac+multiple glu</b>: 93% (n = 1/14) compared with <b>ac+1xglu</b>: 48% (n = 10/21) (p<0.02). No significant difference in survival rate between <b>ac+multiple glu</b>: 93% (n = 13/14) and <b>glu</b>: 77% (n = 17/22).</p