The influence of paraventricular nucleus of the hypothalamus soluble guanylate cyclase on autonomic and neuroendocrine responses to acute restraint stress in rats.

The paraventricular nucleus of the hypothalamus (PVN) regulates physiological and behavioural responses evoked by stressful stimuli, but the local neurochemical and signalling mechanisms involved are not completely understood. The soluble guanylate cyclase (sGC) within the PVN is implicated in autonomic and cardiovascular control in rodents under resting conditions. However, the involvement of PVN sGC-mediated signalling in stress responses is unknown. Therefore, we investigated the role of sGC within the PVN in cardiovascular, autonomic, neuroendocrine, and local neuronal responses to acute restraint stress in rats. Bilateral microinjection of the selective sGC inhibitor ODQ (1 nmol/100 nl) into the PVN reduced both the increased arterial pressure and the drop in cutaneous tail temperature evoked by restraint stress, while the tachycardia was enhanced. Intra-PVN injection of ODQ did not alter the number of Fos-immunoreactive neurons in either the dorsal cap parvocellular (PaDC), ventromedial (PaV), medial parvocellular (PaMP), or lateral magnocelllular (PaLM) portions of the PVN following acute restraint stress. Local microinjection of ODQ into the PVN did not affect the restraint-induced increases in plasma corticosterone concentration. Taken together, these findings suggest that sGC-mediated signalling in the PVN plays a key role in acute stress-induced pressor responses and sympathetically mediated cutaneous vasoconstriction, whereas the tachycardiac response is inhibited. Absence of an effect of ODQ on corticosterone and PVN neuronal activation in and the PaV and PaMP suggests that PVN sGC is not involved in restraint-evoked hypothalamus-pituitary-adrenal (HPA) axis activation and further indicates that autonomic and neuroendocrine responses are dissociable at the level of the PVN.

The glucocorticoid receptor specific modulator CORT108297 reduces brain pathology following status epilepticus.

OBJECTIVE: Glucocorticoid levels rise rapidly following status epilepticus and remain elevated for weeks after the injury. To determine whether glucocorticoid receptor activation contributes to the pathological sequelae of status epilepticus, mice were treated with a novel glucocorticoid receptor modulator, C108297. METHODS: Mice were treated with either C108297 or vehicle for 10 days beginning one day after pilocarpine-induced status epilepticus. Baseline and stress-induced glucocorticoid secretion were assessed to determine whether hypothalamic-pituitary-adrenal axis hyperreactivity could be controlled. Status epilepticus-induced pathology was assessed by quantifying ectopic hippocampal granule cell density, microglial density, astrocyte density and mossy cell loss. Neuronal network function was examined indirectly by determining the density of Fos immunoreactive neurons following restraint stress. RESULTS: Treatment with C108297 attenuated corticosterone hypersecretion after status epilepticus. Treatment also decreased the density of hilar ectopic granule cells and reduced microglial proliferation. Mossy cell loss, on the other hand, was not prevented in treated mice. C108297 altered the cellular distribution of Fos protein but did not restore the normal pattern of expression. INTERPRETATION: Results demonstrate that baseline corticosterone levels can be normalized with C108297, and implicate glucocorticoid signaling in the development of structural changes following status epilepticus. These findings support the further development of glucocorticoid receptor modulators as novel therapeutics for the prevention of brain pathology following status epilepticus.

Prefrontal cortex PACAP signaling: organization and role in stress regulation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an excitatory neuromodulatory peptide strongly implicated in nervous stress processing. Human polymorphism of the primary PACAP receptor (PAC1) is linked to psychiatric disorders, including posttraumatic stress disorder (PTSD). Prefrontal cortex PACAP signaling is associated with processing of traumatic stress and fear learning, suggesting a potential role in PTSD-related deficits. We used RNAscope to define the cellular location of PACAP and PAC1 in the infralimbic cortex (IL). Subsequent experiments used a pharmacological approach to assess the impact of IL PACAP infusion on behavioral and physiological stress response and fear memory. Adult male Sprague-Dawley rats were bilaterally microinjected with PACAP (1 ug) or vehicle into the IL, 30 minutes prior to forced swim test (FST). Blood was sampled at 15, 30, 60, and 120 minutes for analysis of hypothalamic pituitary adrenal (HPA) axis reactivity. Five days after, animals were tested in a 3-day passive avoidance paradigm with subsequent testing of fear retention two weeks later. We observed that PACAP is highly expressed in putative pyramidal neurons (identified by VGlut1 expression), while PAC1 is enriched in interneurons (identified by GAD). Pretreatment with PACAP increased active coping style in the FST, despite higher levels of ACTH and corticosterone. The treatment was also sufficient to cause an increase in anxiety-like behavior in a dark/light crossover test and enhanced retention of passive avoidance. Our data suggest that IL PACAP plays a role in driving stress responses and in processing of fear memories, likely mediated by inhibition of cortical drive.

Chemogenetic Inhibition of Infralimbic Prefrontal Cortex GABAergic Parvalbumin Interneurons Attenuates the Impact of Chronic Stress in Male Mice.

Hypofunction of the prefrontal cortex (PFC) contributes to stress-related neuropsychiatric illnesses. Mechanisms leading to prefrontal hypoactivity remain to be determined. Prior evidence suggests that chronic stress leads to an increase in activity of parvalbumin (PV) expressing GABAergic interneurons (INs) in the PFC. The purpose of the study was to determine whether reducing PV IN activity in the Infralimbic (IL) PFC would prevent stress-related phenotypes. We used a chemogenetic approach to inhibit IL PFC PV INs during stress. Mice were first tested in the tail suspension test (TST) to determine the impact of PV IN inhibition on behavioral responses to acute stress. The long-term impact of PV IN inhibition during a modified chronic variable stress (CVS) was tested in the forced swim test (FST). Acute PV IN inhibition reduced active (struggling) and increased passive coping behaviors (immobility) in the TST. In contrast, inhibition of PV INs during CVS increased active and reduced passive coping behaviors in the FST. Moreover, chronic inhibition of PV INs attenuated CVS-induced changes in Fos expression in the prelimbic cortex (PrL), basolateral amygdala (BLA), and ventrolateral periaqueductal gray (vlPAG) and also attenuated adrenal hypertrophy and body weight loss associated with chronic stress. Our results suggest differential roles of PV INs in acute versus chronic stress, indicative of distinct biological mechanisms underlying acute versus chronic stress responses. Our results also indicate a role for PV INs in driving chronic stress adaptation and support literature evidence suggesting cortical GABAergic INs as a therapeutic target in stress-related illnesses.

Lasting Impact of Chronic Adolescent Stress and Glucocorticoid Receptor Selective Modulation in Male and Female Rats.

Adolescent animals are vulnerable to the effects of stress on brain development. We hypothesized that long-term effects of adolescent chronic stress are mediated by glucocorticoid receptor (GR) signaling. We used a specific GR modulator (CORT108297) to pharmacologically disrupt GR signaling in adolescent rats during exposure to chronic variable stress (CVS). Male and female rats received 30 mg/kg of drug during a 2-week CVS protocol starting at PND46. Emotional reactivity (open field) and coping behaviors (forced swim test (FST)) were then tested in adulthood, 5 weeks after the end of the CVS protocol. Blood samples were collected two days before FST and serial samples after the onset of the swim test to determine baseline and stress response levels of HPA hormones respectively. Our results support differential behavioral, physiological and stress circuit reactivity to adolescent chronic stress exposure in males and females, with variable involvement of GR signaling. In response to adolescent stress, males had heightened reactivity to novelty and exhibited marked reduction in neuronal excitation following swim stress in adulthood, whereas females developed a passive coping strategy in the FST and enhanced HPA axis stress reactivity. Only the latter effect was attenuated by treatment with the GR modulator C108297. In summary, our data suggest that adolescent stress differentially affects emotional behavior and circuit development in males and females, and that GR manipulation during stress can reverse at least some of these effects.

Prefrontal Cortex Regulates Chronic Stress-Induced Cardiovascular Susceptibility.

Background The medial prefrontal cortex is necessary for appropriate appraisal of stressful information, as well as coordinating visceral and behavioral processes. However, prolonged stress impairs medial prefrontal cortex function and prefrontal-dependent behaviors. Additionally, chronic stress induces sympathetic predominance, contributing to health detriments associated with autonomic imbalance. Previous studies identified a subregion of rodent prefrontal cortex, infralimbic cortex (IL), as a key regulator of neuroendocrine-autonomic integration after chronic stress, suggesting that IL output may prevent chronic stress-induced autonomic imbalance. In the current study, we tested the hypothesis that the IL regulates hemodynamic, vascular, and cardiac responses to chronic stress. Methods and Results A viral-packaged small interfering RNA construct was used to knockdown vesicular glutamate transporter 1 (vGluT1) and reduce glutamate packaging and release from IL projection neurons. Male rats were injected with a vGluT1 small interfering RNA-expressing construct or GFP (green fluorescent protein) control into the IL and then remained as unstressed controls or were exposed to chronic variable stress. IL vGluT1 knockdown increased heart rate and mean arterial pressure reactivity, while chronic variable stress increased chronic mean arterial pressure only in small interfering RNA-treated rats. In another cohort, chronic variable stress and vGluT1 knockdown interacted to impair both endothelial-dependent and endothelial-independent vasoreactivity ex vivo. Furthermore, vGluT1 knockdown and chronic variable stress increased histological markers of fibrosis and hypertrophy. Conclusions Knockdown of glutamate release from IL projection neurons indicates that these cells are necessary to prevent the enhanced physiological responses to stress that promote susceptibility to cardiovascular pathophysiology. Ultimately, these findings provide evidence for a neurobiological mechanism mediating the relationship between stress and poor cardiovascular health outcomes.

Conditional deletion of glucocorticoid receptors in rat brain results in sex-specific deficits in fear and coping behaviors.

Glucocorticoid receptors (GR) have diverse functions relevant to maintenance of homeostasis and adaptation to environmental challenges. Understanding the importance of tissue-specific GR function in physiology and behavior has been hampered by near-ubiquitous localization in brain and body. Here we use CRISPR/Cas9 gene editing to create a conditional GR knockdown in Sprague Dawley rats. To test the impact of cell- and region-specific GR knockdown on physiology and behavior, we targeted GR knockdown to output neurons of the prelimbic cortex. Prelimbic knockdown of GR in females caused deficits in acquisition and extinction of fear memory during auditory fear conditioning, whereas males exhibited enhanced active-coping behavior during forced swim. Our data support the utility of this conditional knockdown rat to afford high-precision knockdown of GR across a variety of contexts, ranging from neuronal depletion to circuit-wide manipulations, leveraging the behavioral tractability and enhanced brain size of the rat as a model organism.

Nitrergic neurotransmission in the paraventricular nucleus of the hypothalamus modulates autonomic, neuroendocrine and behavioral responses to acute restraint stress in rats.

We investigated the involvement of nitrergic neurotransmission within the paraventricular nucleus of the hypothalamus (PVN) in modulation of local neuronal activation, autonomic and neuroendocrine responses and behavioral consequences of acute restraint stress in rats. Bilateral microinjections of the selective neuronal nitric oxide (NO) synthase (nNOS) inhibitor Nw-Propyl-L-arginine (NPLA) or the NO scavenger carboxy-PTIO into the PVN reduced arterial pressure and heart rate increases, as well as the fall in cutaneous tail temperature induced by restraint stress. PVN injection of either NPLA or carboxy-PTIO also inhibited restraint-induced increases in anxiety-related behaviors in the elevated plus-maze 24 h later. Local microinjection of NPLA or carboxy-PTIO into the PVN reduced the number of c-fos-immunoreactive neurons in the dorsal parvocellular, ventromedial, medial parvocellular and lateral magnocelllular portions of the PVN in animals subjected to restraint stress. However, neither NPLA nor carboxy-PTIO into the PVN affected restraint-induced increases in plasma corticosterone concentration. The present results indicate that PVN nitrergic neurotransmission acting via nNOS activation has a facilitatory influence on autonomic responses to acute restraint and the delayed emotional consequences of restraint stress. Our results also provide evidence of a prominent role of local nitrergic neurotransmission in PVN neuronal activation during stress.

Functional disruption of stress modulatory circuits in a model of temporal lobe epilepsy.

Clinical data suggest that the neuroendocrine stress response is chronically dysregulated in a subset of patients with temporal lobe epilepsy (TLE), potentially contributing to both disease progression and the development of psychiatric comorbidities such as anxiety and depression. Whether neuroendocrine dysregulation and psychiatric comorbidities reflect direct effects of epilepsy-related pathologies, or secondary effects of disease burden particular to humans with epilepsy (i.e. social estrangement, employment changes) is not clear. Animal models provide an opportunity to dissociate these factors. Therefore, we queried whether epileptic mice would reproduce neuroendocrine and behavioral changes associated with human epilepsy. Male FVB mice were exposed to pilocarpine to induce status epilepticus (SE) and the subsequent development of spontaneous recurrent seizures. Morning baseline corticosterone levels were elevated in pilocarpine treated mice at 1, 7 and 10 weeks post-SE relative to controls. Similarly, epileptic mice had increased adrenal weight when compared to control mice. Exposure to acute restraint stress resulted in hypersecretion of corticosterone 30 min after the onset of the challenge. Anatomical analyses revealed reduced Fos expression in infralimbic and prelimbic prefrontal cortex, ventral subiculum and basal amygdala following restraint. No differences in Fos immunoreactivity were found in the paraventricular nucleus of the hypothalamus, hippocampal subfields or central amygdala. In order to assess emotional behavior, a second cohort of mice underwent a battery of behavioral tests, including sucrose preference, open field, elevated plus maze, 24h home-cage monitoring and forced swim. Epileptic mice showed increased anhedonic behavior, hyperactivity and anxiety-like behaviors. Together these data demonstrate that epileptic mice develop HPA axis hyperactivity and exhibit behavioral dysfunction. Endocrine and behavioral changes are associated with impaired recruitment of forebrain circuits regulating stress inhibition and emotional reactivity. Loss of forebrain control may underlie pronounced endocrine dysfunction and comorbid psychopathologies seen in temporal lobe epilepsy.

Desacyl Ghrelin Decreases Anxiety-like Behavior in Male Mice.

Ghrelin is a 28-amino acid polypeptide that regulates feeding, glucose metabolism, and emotionality (stress, anxiety, and depression). Plasma ghrelin circulates as desacyl ghrelin (DAG) or, in an acylated form, acyl ghrelin (AG), through the actions of ghrelin O-acyltransferase (GOAT), exhibiting low or high affinity, respectively, for the growth hormone secretagogue receptor (GHSR) 1a. We investigated the role of endogenous AG, DAG, and GHSR1a signaling on anxiety and stress responses using ghrelin knockout (Ghr KO), GOAT KO, and Ghsr stop-floxed (Ghsr null) mice. Behavioral and hormonal responses were tested in the elevated plus maze and light/dark (LD) box. Mice lacking both AG and DAG (Ghr KO) increased anxiety-like behaviors across tests, whereas anxiety reactions were attenuated in DAG-treated Ghr KO mice and in mice lacking AG (GOAT KO). Notably, loss of GHSR1a (Ghsr null) did not affect anxiety-like behavior in any test. Administration of AG and DAG to Ghr KO mice with lifelong ghrelin deficiency reduced anxiety-like behavior and decreased phospho-extracellular signal-regulated kinase phosphorylation in the Edinger-Westphal nucleus in wild-type mice, a site normally expressing GHSR1a and involved in stress- and anxiety-related behavior. Collectively, our data demonstrate distinct roles for endogenous AG and DAG in regulation of anxiety responses and suggest that the behavioral impact of ghrelin may be context dependent.

Chronic Stress Increases Prefrontal Inhibition: A Mechanism for Stress-Induced Prefrontal Dysfunction.

BACKGROUND: Multiple neuropsychiatric disorders, e.g., depression, are linked to imbalances in excitatory and inhibitory neurotransmission and prefrontal cortical dysfunction, and are concomitant with chronic stress. METHODS: We used electrophysiologic (n = 5-6 animals, 21-25 cells/group), neuroanatomic (n = 6-8/group), and behavioral (n = 12/group) techniques to test the hypothesis that chronic stress increases inhibition of medial prefrontal cortex (mPFC) glutamatergic output neurons. RESULTS: Using patch clamp recordings from infralimbic mPFC pyramidal neurons, we found that chronic stress selectively increases the frequency of miniature inhibitory postsynaptic currents with no effect on amplitude, which suggests that chronic stress increases presynaptic gamma-aminobutyric acid release. Elevated gamma-aminobutyric acid release under chronic stress is accompanied by increased inhibitory appositions and terminals onto glutamatergic cells, as assessed by both immunohistochemistry and electron microscopy. Furthermore, chronic stress decreases glucocorticoid receptor immunoreactivity specifically in a subset of inhibitory neurons, which suggests that increased inhibitory tone in the mPFC after chronic stress may be caused by loss of a glucocorticoid receptor-mediated brake on interneuron activity. These neuroanatomic and functional changes are associated with impairment of a prefrontal-mediated behavior. During chronic stress, rats initially make significantly more errors in the delayed spatial win-shift task, an mPFC-mediated behavior, which suggests a diminished impact of the mPFC on decision making. CONCLUSIONS: Taken together, the data suggest that chronic stress increases synaptic inhibition onto prefrontal glutamatergic output neurons, limiting the influence of the prefrontal cortex in control of stress reactivity and behavior. Thus, these data provide a mechanistic link among chronic stress, prefrontal cortical hypofunction, and behavioral dysfunction.

Divergent effects of repeated restraint versus chronic variable stress on prefrontal cortical immune status after LPS injection.

Previous work from our group has shown that chronic homotypic stress (repeated restraint - RR) increases microglial morphological activation in the prefrontal cortex (PFC), while chronic heterotypic stress (chronic variable stress - CVS) produces no such effect. Therefore, we hypothesized that stressor modality would also determine the susceptibility of the PFC to a subsequent inflammatory stimulus (low dose lipopolysaccharide (LPS)). We found that RR, but not CVS, increased Iba-1 soma size in the PFC after LPS injection, consistent with microglial activation. In contrast, CVS decreased gene expression of proinflammatory cytokines and Iba-1 in the PFC under baseline conditions, which were not further affected by LPS. Thus, RR appears to promote microglial responses to LPS, whereas CVS is largely immunosuppressive. The results suggest that neuroimmune changes caused by CVS may to some extent protect the PFC from subsequent inflammatory stimuli. These data suggest that modality and/or intensity of stressful experiences will be a major determinant of central inflammation and its effect on prefrontal cortex-mediated functions.

Adolescent chronic stress causes hypothalamo-pituitary-adrenocortical hypo-responsiveness and depression-like behavior in adult female rats.

Adolescence is a period of substantial neuroplasticity in stress regulatory neurocircuits. Chronic stress exposure during this period leads to long-lasting changes in neuroendocrine function and emotional behaviors, suggesting adolescence may be a critical period for development of stress vulnerability. This study investigated the effects of exposure to 14 days of chronic variable stress (CVS) in late-adolescent (pnd 45-58) female rats on neuroendocrine function, neuropeptide mRNA expression and depressive-like behavior in adolescence (pnd 59) and in adulthood (pnd 101). Adult females exposed to CVS in adolescence have a blunted hypothalamo-pituitary-adrenocortical (HPA) axis in response to a novel stressor and increased immobility in the forced swim test. Blunted HPA axis responses were accompanied by reduced vasopressin mRNA expression in the paraventricular nucleus of the hypothalamus (PVN), suggesting decreased central drive. Adolescent females tested immediately after CVS did not exhibit differences in stress reactivity or immobility in the forced swim test, despite evidence for enhanced central HPA axis drive (increased CRH mRNA expression in PVN). Overall, our study demonstrates that exposure to chronic stress in adolescence is sufficient to induce lasting changes in neuroendocrine drive and behavior, potentially altering the developmental trajectory of stress circuits as female rats age into adulthood.

Role of nucleus of the solitary tract noradrenergic neurons in post-stress cardiovascular and hormonal control in male rats.

Chronic stress causes hypothalamo-pituitary-adrenal (HPA) axis hyperactivity and cardiovascular dyshomeostasis. Noradrenergic (NA) neurons in the nucleus of the solitary tract (NTS) are considered to play a role in these changes. In this study, we tested the hypothesis that NTS NA A2 neurons are required for cardiovascular and HPA axis responses to both acute and chronic stress. Adult male rats received bilateral microinjection into the NTS of 6-hydroxydopamine (6-OHDA) to lesion A2 neurons [cardiovascular study, n = 5; HPA study, n = 5] or vehicle [cardiovascular study, n = 6; HPA study, n = 4]. Rats were exposed to acute restraint stress followed by 14 d of chronic variable stress (CVS). On the last day of testing, rats were placed in a novel elevated plus maze (EPM) to test post-CVS stress responses. Lesions of NTS A2 neurons reduced the tachycardic response to acute restraint, confirming that A2 neurons promote sympathetic activation following acute stress. In addition, CVS increased the ratio of low-frequency to high-frequency power for heart rate variability, indicative of sympathovagal imbalance, and this effect was significantly attenuated by 6-OHDA lesion. Lesions of NTS A2 neurons reduced acute restraint-induced corticosterone secretion, but did not affect the corticosterone response to the EPM, indicating that A2 neurons promote acute HPA axis responses, but are not involved in CVS-mediated HPA axis sensitization. Collectively, these data indicate that A2 neurons promote both cardiovascular and HPA axis responses to acute stress. Moreover, A2 catecholaminergic neurons may contribute to the potentially deleterious enhancement of sympathetic drive following chronic stress.

Glucocorticoid regulation of preproglucagon transcription and RNA stability during stress.

Stress elicits a synchronized response of the endocrine, sympathetic, and central nervous systems to preserve homeostasis and well-being. Glucagon-like peptide-1 (GLP-1), a primary posttranslational product of the preproglucagon (PPG) gene, activates both physical and psychological stress responses. The current study examined mechanisms regulating expression of PPG gene products in the hindbrain. Our results indicate that PPG mRNA decreases rapidly after exposure to acute stressors of multiple modalities. Reduced mRNA levels are accompanied by reduced GLP-1 immunoreactivity in the paraventricular nucleus of hypothalamus, suggesting release at PPG terminals. Stress-induced decrements in PPG mRNA were attenuated in adrenalectomized-corticosterone-replaced rats, suggesting that mRNA down-regulation is due at least in part to glucocorticoid secretion. In contrast, acute stress increased levels of PPG heteronuclear RNA (hnRNA) in a glucocorticoid-dependent manner, suggesting that decreases in PPG mRNA are due to increased degradation rather than reduced transcription. Glucocorticoid administration to unstressed rats is sufficient to cause decrements in PPG mRNA and increments in PPG hnRNA. These findings suggest that glucocorticoids deplete the pool of transcribed PPG mRNA and concurrently stimulate PPG gene transcription, with the latter allowing a mechanism for replenishment of PPG mRNA after stress cessation. The combination of rapid PPG mRNA depletion and initiation of PPG transcription within 30 min is consistent with a rapid action of glucocorticoids on GLP-1 bioavailability, resulting in a transient reduction in the capacity for neuropeptidergic excitation of stress responses.

Daily limited access to sweetened drink attenuates hypothalamic-pituitary-adrenocortical axis stress responses.

Stress can promote palatable food intake, and consumption of palatable foods may dampen psychological and physiological responses to stress. Here we develop a rat model of daily limited sweetened drink intake to further examine the linkage between consumption of preferred foods and hypothalamic-pituitary-adrenocortical axis responses to acute and chronic stress. Adult male rats with free access to water were given additional twice-daily access to 4 ml sucrose (30%), saccharin (0.1%; a noncaloric sweetener), or water. After 14 d of training, rats readily learned to drink sucrose and saccharin solutions. Half the rats were then given chronic variable stress (CVS) for 14 d immediately after each drink exposure; the remaining rats (nonhandled controls) consumed their appropriate drinking solution at the same time. On the morning after CVS, responses to a novel restraint stress were assessed in all rats. Multiple indices of chronic stress adaptation were effectively altered by CVS. Sucrose consumption decreased the plasma corticosterone response to restraint stress in CVS rats and nonhandled controls; these reductions were less pronounced in rats drinking saccharin. Sucrose or saccharin consumption decreased CRH mRNA expression in the paraventricular nucleus of the hypothalamus. Moreover, sucrose attenuated restraint-induced c-fos mRNA expression in the basolateral amygdala, infralimbic cortex, and claustrum. These data suggest that limited consumption of sweetened drink attenuates hypothalamic-pituitary-adrenocortical axis stress responses, and calories contribute but are not necessary for this effect. Collectively the results support the hypothesis that the intake of palatable substances represents an endogenous mechanism to dampen physiological stress responses.

Plasma infusions into porcine cerebral white matter induce early edema, oxidative stress, pro-inflammatory cytokine gene expression and DNA fragmentation: implications for white matter injury with increased blood-brain-barrier permeability.

Plasma infused into porcine cerebral white matter induces both acute interstitial and delayed vasogenic edema. Edematous white matter contains extracellular plasma proteins and rapidly induces oxidative stress as evidenced by increased protein carbonyl formation and heme oxygenase-1 induction. We tested the hypothesis that edematous white matter would also upregulate pro-inflammatory cytokine gene expression and develop DNA damage. We infused autologous plasma into the frontal hemispheric white matter of pentobarbital-anesthetized pigs. We monitored and controlled physiological variables and froze brains in situ at 1, 4 or 24 hrs. We determined edema volumes by computer-assisted morphometry. We measured white matter protein carbonyl formation by immunoblotting, cytokine gene expression by standard RT-PCR methods and DNA fragmentation by agarose gel electrophoresis. White matter edema developed acutely (1 hr) after plasma infusion and increased significantly in volume between 4 and 24 hrs. Protein carbonyl formation also occurred rapidly in edematous white matter with significant elevations (3 to 4-fold) already present at 1 hr. This increase remained through 24 hrs. Pro-inflammatory cytokine gene expression was also rapidly increased at 1 hr post-infusion. Evidence for DNA fragmentation began at 2 to 4 hrs, and a pattern indicative of both ongoing necrosis and apoptosis was robust by 24 hrs. Plasma protein accumulation in white matter induces acute edema development and a cascade of patho-chemical events including oxidative stress, pro-inflammatory cytokine gene expression and DNA damage. These results suggest that in diseases with increased blood-brain barrier (BBB) permeability or following intracerebral hemorrhage or traumatic brain injury, interstitial plasma can rapidly damage white matter.

Protein oxidation and heme oxygenase-1 induction in porcine white matter following intracerebral infusions of whole blood or plasma.

Spontaneous or traumatic intracerebral hemorrhage (ICH) in the white matter of neonates, children and adults causes significant mortality and morbidity. The detailed biochemical mechanisms through which blood damages white matter are poorly defined. Presently, we tested the hypothesis that ICH induces rapid oxidative stress in white matter. Also, since clot-derived plasma proteins accumulate in white matter after ICH and these proteins can induce oxidative stress in microglia in vitro, we determined whether the blood's plasma component alone induces oxidative stress. Lastly, since heme oxygenase-1 (HO-1) induction is highly sensitive to oxidative stress, we also examined white matter HO-1 gene expression. We infused either whole blood or plasma (2.5 ml) into the frontal hemispheric white matter of pentobarbital-anesthetized pigs ( approximately 1 kg) over 15 min. We monitored and controlled physiologic variables and froze brains in situ between 1 and 24 h after ICH. White matter oxidative stress was determined by measuring protein carbonyl formation and HO-1 gene expression by RT-PCR. Protein carbonyl formation occurred rapidly in the white matter adjacent to both blood and plasma clots with significant elevations (3- to 4-fold) already 1 h after infusion. This increase remained through the first 24 h. HO-1 mRNA was rapidly induced in white matter with either whole blood or plasma infusions. These results demonstrate that not only whole blood but also its plasma component are capable of rapidly inducing oxidative stress in white matter. This rapid response, possibly in microglial cells, may contribute to white matter damage not only following ICH, but also in pathophysiological states in which blood-brain-barrier permeability to plasma proteins is increased.