Robert M. Brucklacher

Experimental stroke in the female diabetic, db/db, mouse

Diabetic hyperglycemia increases brain damage after cerebral ischemia in animals and humans, although the underlying mechanisms remain unclear. Gender-linked differences in ischemic tolerance have been described but have not been studied in the context of diabetes. In the current study, we used a model of unilateral common carotid artery ligation, combined with systemic hypoxia, to study the effects of diabetes and gender on hypoxic–ischemic (HI) brain damage in the genetic model of Type II diabetes, the db/db, mouse. Male and female, control and db/db, mice were subjected to right common carotid artery ligation followed by varying periods of hypoxia (8% oxygen/92% nitrogen) to assess mortality, infarct volume, and tissue damage by light microscopic techniques. End-ischemic regional cerebral blood flow (CBF) was determined using [14C] iodoantipyrine autoradiography. Glycolytic and high energy phosphate compounds were measured in blood and brain by enzymatic and fluorometric techniques. Gender and diabetes had significant effects on mortality from HI and extent of brain damage in the survivors. Female mice were more resistant than their male counterparts, such that the severity (mortality and infarction size) in the male diabetics > female diabetics ~ male controls > female controls. End-ischemic CBF and depletion of cerebral high energy reserves were comparable among all groups. Surprisingly, female diabetic mice were more hyperglycemic and demonstrated a greater prolonged lactacidosis than the males; however, they were more resistant to damage. The results suggest a unique pathophysiology of hypoxia–ischemia in the female diabetic brain.

Effect of Carbon Dioxide on Cerebral Metabolism during Hypoxia-Ischemia in the Immature Rat

We previously have demonstrated that hypocapnia aggravates and hypercapnia protects the immature rat from hypoxicischemic brain damage. To ascertain cerebral blood flow (CBF) and metabolic correlates, 7-d postnatal rats were subjected to hypoxia-ischemia during which they were rendered either hypo-(3.5 kPa), normo- (5.1 kPa), or hypercapnic (7.3 kPa) by the inhalation of either 0, 3, or 6% CO2, 8% O2, balance N2. CBF during hypoxia-ischemia was better preserved in the normo- and hypercapnic rat pups; these animals also exhibited a stimulation of cerebral glucose utilization. Brain glucose concentrations were higher and lactate lower in the normo- and hypercapnic animals, indicating that glucose was consumed oxidatively in these groups rather than by anaerobic glycolysis, as apparently occurred in the hypocapnic animals. ATP and phosphocreatine were better preserved in the normo- and hypercapnic rats compared with the hypocapnic animals. Cerebrospinal fluid glutamate, as a reflection of the brain extracellular fluid concentration, was lowest in the hypercapnic rats at 2 h of hypoxia-ischemia. The data indicate that during hypoxia-ischemia in the immature rat, CBF is better preserved during normo- and hypercapnia; the greater oxygen delivery promotes cerebral glucose utilization and oxidative metabolism for optimal maintenance of tissue high energy phosphate reserves. An inhibition of glutamate secretion into the synaptic cleft and its attenuation of N-methyl-D-aspartate receptor activation would further protect the hypercapnic animal from hypoxic-ischemic brain damage.

Carbohydrate and Energy Metabolism during the Evolution of Hypoxic-Ischemic Brain Damage in the Immature Rat

The brain damage that evolves from perinatal cerebral hypoxia-ischemia may involve lingering disturbances in metabolic activity that proceed into the recovery period. To clarify this issue, we determined the carbohydrate and energy status of cerebral tissue using enzymatic, fluorometric techniques in an experimental model of perinatal hypoxic-ischemic brain damage. Seven-day postnatal rats were subjected to unilateral common carotid artery ligation followed by 3 h of hypoxia with 8% oxygen at 37°C. This insult is known to produce tissue injury (selective neuronal necrosis or infarction) predominantly in the cerebral hemisphere ipsilateral to the carotid artery occlusion in 92% of the animals. Rat pups were quick-frozen in liquid nitrogen at 0, 1, 4, 12, 24, or 72 h of recovery; littermate controls underwent neither ligation nor hypoxia. Glucose in both cerebral hemispheres was nearly completely exhausted during hypoxia-ischemia, with concurrent increases in lactate to 10 mmol/kg. During recovery, glucose promptly increased above control values, suggesting an inhibition of glycolytic flux, as documented in the ipsilateral cerebral hemisphere by measurement of glucose utilization (CMRglc) at 24 h. Tissue lactate declined rapidly during recovery but remained slightly elevated in the ipsilateral hemisphere for 12 h. Phosphocreatine (P∼Cr) and ATP in the ipsilateral cerebral hemisphere were 14 and 26% of control (p < 0.001) at the end of hypoxia-ischemia; total adenine nucleotides (ATP + ADP + AMP) also were partially depleted (–46%). During the first hour of recovery, mean P∼Cr was replenished to within 90% of baseline, whereas mean ATP was incompletely restored to 68–81% of control (p < 0.05). Individual ATP and total adenine nucleotide values were >2 SD below control levels in 17/24 (71%) brains at all intervals of recovery. Both ATP and total adenine nucleotides were inversely correlated with tissue water content, reflecting the extent of cerebral edema. No major alterations in the high-energy phosphate reserves occurred in the contralateral cerebral hemisphere either during or following hypoxia-ischemia. Thus, following perinatal cerebral hypoxia-ischemia, ATP and total adenine nucleotides never recover completely in brains undergoing damage but rather are permanently depleted to levels that reflect the severity of tissue injury. Recovery of P∼Cr to near normal levels can occur despite evolving brain damage. The findings have relevance to the assessment of asphyxiated newborn humans using magnetic resonance spectroscopy.

Persistent alterations in mesolimbic gene expression with abstinence from cocaine self-administration

Cocaine-responsive gene expression changes have been described after either no drug abstinence or short periods of abstinence. Little data exist on the persistence of these changes after long-term abstinence. Previously, we reported that after discrete-trial cocaine self-administration and 10 days of forced abstinence, incubation of cocaine reinforcement was observable by a progressive ratio schedule. The present study used rat discrete-trial cocaine self-administration and long-term forced abstinence to examine extinction responding, mRNA abundance of known cocaine-responsive genes, and chromatin remodeling. At 30 and 100 days of abstinence, extinction responding increased compared to 3-day abstinent rats. Decreases in both medial prefrontal cortex (mPFC) and nucleus accumbens c-fos, Nr4a1, Arc, and EGR1 mRNA were observed, and in most cases persisted, for 100 days of abstinence. The signaling peptides CART and neuropeptide Y (NPY) transiently increased in the mPFC, but returned to baseline levels following 10 days of abstinence. To investigate a potential regulatory mechanism for these persistent mRNA changes, levels of histone H3 acetylation at promoters for genes with altered mRNA expression were examined. In the mPFC, histone H3 acetylation decreased after 1 and 10 days of abstinence at the promoter for EGR1. H3 acetylation increased for NPY after 1 day of abstinence and returned to control levels by 10 days of abstinence. Behaviorally, these results demonstrate incubation after discrete-trial cocaine self-administration and prolonged forced abstinence. This incubation is accompanied by changes in gene expression that persist long after cessation of drug administration and may be regulated by chromatin remodeling.

Whole genome assessment of the retinal response to diabetes reveals a progressive neurovascular inflammatory response

BackgroundDespite advances in the understanding of diabetic retinopathy, the nature and time course of molecular changes in the retina with diabetes are incompletely described. This study characterized the functional and molecular phenotype of the retina with increasing durations of diabetes.ResultsUsing the streptozotocin-induced rat model of diabetes, levels of retinal permeability, caspase activity, and gene expression were examined after 1 and 3 months of diabetes. Gene expression changes were identified by whole genome microarray and confirmed by qPCR in the same set of animals as used in the microarray analyses and subsequently validated in independent sets of animals. Increased levels of vascular permeability and caspase-3 activity were observed at 3 months of diabetes, but not 1 month. Significantly more and larger magnitude gene expression changes were observed after 3 months than after 1 month of diabetes. Quantitative PCR validation of selected genes related to inflammation, microvasculature and neuronal function confirmed gene expression changes in multiple independent sets of animals.ConclusionThese changes in permeability, apoptosis, and gene expression provide further evidence of progressive retinal malfunction with increasing duration of diabetes. The specific gene expression changes confirmed in multiple sets of animals indicate that pro-inflammatory, anti-vascular barrier, and neurodegenerative changes occur in tandem with functional increases in apoptosis and vascular permeability. These responses are shared with the clinically documented inflammatory response in diabetic retinopathy suggesting that this model may be used to test anti-inflammatory therapeutics.

Diabetes downregulates presynaptic proteins and reduces basal synapsin I phosphorylation in rat retina

Diabetic retinopathy can result in vision loss and involves progressive neurovascular degeneration of the retina. This study tested the hypothesis that diabetes decreases the retinal expression of presynaptic proteins involved in synaptic function. The protein and mRNA contents for synapsin I, synaptophysin, vesicle‐associated membrane protein 2, synaptosomal‐associated protein of 25 kDa and postsynaptic density protein of 95 kDa were measured by immunohistochemistry, immunoblotting and real‐time quantitative polymerase chain reaction in whole retinas and retinal synaptosomes from streptozotocin‐diabetic and control Sprague–Dawley rats. There was less presynaptic protein immunoreactivity after 1 and 3 months of diabetes than in controls. Discrete synaptophysin‐immunoreactive puncta were significantly smaller and fewer in sections from 1‐ and 3‐month diabetic rat retinas than in those from controls. The content of presynaptic proteins was significantly less in whole retinas of 1‐ and 3‐month diabetic rats, and in synaptosomes from 1‐month diabetic rats, than in controls. Whole retinas had significantly less mRNA for these genes after 3 months but not 1 month of diabetes, as compared to controls (with the exception of postsynaptic density protein of 95 kDa). In contrast, there was significantly less mRNA for synaptic proteins in synaptosomes of 1‐month diabetic rats than in controls, suggesting a localized depletion at synapses. Protein and mRNA for β‐actin and neuron‐specific enolase were unchanged by diabetes. The ratio of phosphorylated to total synapsin I was also reduced in whole retina and isolated synaptosomes from 1‐month diabetic rats, as compared to controls. These data suggest that diabetes has a profound impact on presynaptic protein expression in the retina, and may provide a mechanism for the well‐established defects in vision and the electrophysiological response of the retina in diabetes.

Hypoxic Preconditioning Increases Brain Glycogen and Delays Energy Depletion from Hypoxia-Ischemia in the Immature Rat

Recent studies have shown a protection from cerebral hypoxic-ischemic (HI) brain damage in the immature rat following a prior systemic hypoxic exposure when compared with those not exposed previously. To investigate the mechanism(s) of hypoxic preconditioning, brain glycogen and high-energy phosphate reserves were measured in naïve and preconditioned rat pups subjected to HI. Groups in this study included untouched (naïve) controls, preconditioned controls (i.e., hypoxia only), preconditioned with HI insult, and naïve pups with HI insult. Hypoxic preconditioning was achieved in postnatal-day-6 rats subjected to 8% systemic hypoxia for 2.5 h at 37°C. Twenty-four hours later, they were subjected to unilateral common carotid artery ligation and systemic hypoxia with 8% oxygen at 37°C for 90 min. Animals were allowed to recover from HI for up to 24 h. At specific intervals, animals in each group were frozen in liquid nitrogen for determination of cerebral metabolites. Preconditioned animals showed a significant increase in brain glycogen 24 h following the initial hypoxic exposure, corresponding to the beginning of the HI insult. Measurement at the end of 90 min of HI showed a depletion of high-energy phosphates, ATP and phosphocreatine, in all animals although ATP remained significantly higher in the preconditioned animals. Thus, the energy from increased glycogen following preconditioning slowed high-energy phosphate depletion during HI, thereby allowing for long-term protection.

The Effect of Hyperglycemia on Cerebral Metabolism during Hypoxia-Ischemia in the Immature Rat

Unlike adults, hyperglycemia with circulating glucose concentrations of 25–35 mM/L protects the immature brain from hypoxic–ischemic damage. To ascertain the effect of hyperglycemia on cerebral oxidative metabolism during the course of hypoxia–ischemia, 7-day postnatal rats underwent unilateral common carotid artery ligation followed by exposure to 8% O2 for 2 h at 37°C. Experimental animals received 0.2 cc s.c. 50% glucose at the onset of hypoxia–ischemia, and 0.15 cc 25% glucose 1 h later to maintain blood glucose concentrations at 20–25 mML for 2 h. Control rat pups received equivalent concentrations or volumes of either mannitol or 1 N saline at the same intervals. The cerebral metabolic rate for glucose (CMRglc.) increased from 7.1 (control) to 20.2 μmol 100 g−1 min−1 in hyperglycemic rats during the first hour of hypoxia–ischemia, 79 and 35% greater than the rates for saline- and mannitol-injected animals at the same interval, respectively (p < 0.01). Brain intracellular glucose concentrations were 5.2 and 3.0 mM/kg in the hyperglycemic rat pups at 1 and 2 h of hypoxia–ischemia, respectively; glucose levels were near negligible in mannitol- and saline-treated animals at the same intervals. Brain intracellular lactate concentrations averaged 13.4 and 23.3 mM/kg in hyperglycemic animals at 1 and 2 h of hypoxia–ischemia, respectively, more than twice the concentrations estimated for the saline- and mannitol-treated littermates. Phosphocreatine (PCr) and ATP decreased in all three experimental groups, but were preserved to the greatest extent in hyperglycemic animals. Results indicate that anaerobic glycolytic flux is increased to a greater extent in hyperglycemic immature rats than in normoglycemic littermates subjected to cerebral hypoxia–ischemia, and that the enhanced glycolysis leads to greater intracellular lactate accumulation. Despite cerebral lactosis, energy reserves were better preserved in hyperglycemic animals than in saline-treated controls, thus accounting for the greater resistance of hyperglycemic animals to hypoxic–ischemic brain damage.

Concurrent hippocampal induction of MHC II pathway components and glial activation with advanced aging is not correlated with cognitive impairment.

BackgroundAge-related cognitive dysfunction, including impairment of hippocampus-dependent spatial learning and memory, affects approximately half of the aged population. Induction of a variety of neuroinflammatory measures has been reported with brain aging but the relationship between neuroinflammation and cognitive decline with non-neurodegenerative, normative aging remains largely unexplored. This study sought to comprehensively investigate expression of the MHC II immune response pathway and glial activation in the hippocampus in the context of both aging and age-related cognitive decline.MethodsThree independent cohorts of adult (12-13 months) and aged (26-28 months) F344xBN rats were behaviorally characterized by Morris water maze testing. Expression of MHC II pathway-associated genes identified by transcriptomic analysis as upregulated with advanced aging was quantified by qPCR in synaptosomal fractions derived from whole hippocampus and in hippocampal subregion dissections (CA1, CA3, and DG). Activation of astrocytes and microglia was assessed by GFAP and Iba1 protein expression, and by immunohistochemical visualization of GFAP and both CD74 (Ox6) and Iba1.ResultsWe report a marked age-related induction of neuroinflammatory signaling transcripts (i.e., MHC II components, toll-like receptors, complement, and downstream signaling factors) throughout the hippocampus in all aged rats regardless of cognitive status. Astrocyte and microglial activation was evident in CA1, CA3 and DG of intact and impaired aged rat groups, in the absence of differences in total numbers of GFAP+ astrocytes or Iba1+ microglia. Both mild and moderate microglial activation was significantly increased in all three hippocampal subregions in aged cognitively intact and cognitively impaired rats compared to adults. Neither induction of MHCII pathway gene expression nor glial activation correlated to cognitive performance.ConclusionsThese data demonstrate a novel, coordinated age-related induction of the MHC II immune response pathway and glial activation in the hippocampus, indicating an allostatic shift toward a para-inflammatory phenotype with advancing age. Our findings demonstrate that age-related induction of these aspects of hippocampal neuroinflammation, while a potential contributing factor, is not sufficient by itself to elicit impairment of spatial learning and memory in models of normative aging. Future efforts are needed to understand how neuroinflammation may act synergistically with cognitive-decline specific alterations to cause cognitive impairment.

Effect of extreme hypercapnia on hypoxic-ischemic brain damage in the immature rat

To ascertain the effect of extreme hypercapnia on perinatal hypoxic-ischemic brain damage, 7-d-postnatal rats were exposed to unilateral common carotid artery occlusion followed by hypoxia with 8% oxygen combined with 3, 12, or 15% carbon dioxide (CO2) for 2 h at 37°C. Survivors underwent neuropathologic examination at 30 d of postnatal age, and their brains were characterized as follows: 0 = normal; 1 = mild atrophy; 2 = moderate atrophy; 3 = cystic infarct with external dimensions <3 mm; and 4 = cystic infarct with external dimensions >3 mm. The width of the cerebral hemisphere ipsilateral to the carotid artery occlusion also was determined on a posterior coronal section and compared with that of the contralateral hemisphere to ascertain the severity of cerebral atrophy/cavitation. CO2 tensions averaged 5.08, 11.1, and 13.2 kPa in the 3, 12, and 15% CO2-exposed animals, respectively, during hypoxia-ischemia (HI). Neuropathologic results showed that immature rats exposed to 3 and 12% CO2 had similar severities of brain damage. In contrast, rat pups exposed to HI combined with 15% CO2 were significantly more brain damaged than littermates exposed to 3% CO2. Specifically, eight of 14 animals exposed to 15% CO2 showed cystic infarcts (grades 3 and 4), whereas none of 14 littermates exposed to 3% CO2 developed cystic infarcts (p < 0.01). Analyses of coronal width ratios at each CO2 exposure provided results comparable with those of the gross neuropathology scores. Cerebral blood flow (CBF), measured at 90 min of HI, was lowest in those immature rats exposed to 15% CO2 compared with control (p = 0.04), with higher values in those rat pups exposed to 3 and 12% CO2. The findings indicate that 7-d-postnatal rats exposed to HI with superimposed 12% CO2 are neither less nor more brain damaged than littermates exposed to 3% CO2 (normocapnia). In contrast, animals exposed to 15% CO2 are the most brain damaged of the three groups. Presumably, extreme hypercapnia produces more severe cardiovascular depression than is seen in animals subjected to lesser degrees of hypercapnia; the cardiovascular depression, in turn, leads to greater cerebral ischemia and ultimate brain damage.