Cheryl M. Heesch

Biofabrication and testing of a fully cellular nerve graft

Rupture of a nerve is a debilitating injury with devastating consequences for the individuals quality of life. The gold standard of repair is the use of an autologous graft to bridge the severed nerve ends. Such repair however involves risks due to secondary surgery at the donor site and may result in morbidity and infection. Thus the clinical approach to repair often involves non-cellular solutions, grafts composed of synthetic or natural materials. Here we report on a novel approach to biofabricate fully biological grafts composed exclusively of cells and cell secreted material. To reproducibly and reliably build such grafts of composite geometry we use bioprinting. We test our grafts in a rat sciatic nerve injury model for both motor and sensory function. In particular we compare the regenerative capacity of the biofabricated grafts with that of autologous grafts and grafts made of hollow collagen tubes by measuring the compound action potential (for motor function) and the change in mean arterial blood pressure as consequence of electrically eliciting the somatic pressor reflex. Our results provide evidence that bioprinting is a promising approach to nerve graft fabrication and as a consequence to nerve regeneration.

Hypoxia activates nucleus tractus solitarii neurons projecting to the paraventricular nucleus of the hypothalamus

Peripheral chemoreceptor afferent information is sent to the nucleus tractus solitarii (nTS), integrated, and relayed to other brain regions to alter cardiorespiratory function. The nTS projects to the hypothalamic paraventricular nucleus (PVN), but activation and phenotype of these projections during chemoreflex stimulation is unknown. We hypothesized that activation of PVN-projecting nTS neurons occurs primarily at high intensities of hypoxia. We assessed ventilation and cardiovascular parameters in response to increasing severities of hypoxia. Retrograde tracers were used to label nTS PVN-projecting neurons and, in some rats, rostral ventrolateral medulla (RVLM)-projecting neurons. Immunohistochemistry was performed to identify nTS cells that were activated (Fos-immunoreactive, Fos-IR), catecholaminergic, and GABAergic following hypoxia. Conscious rats underwent 3 h normoxia (n = 4, 21% O(2)) or acute hypoxia (12, 10, or 8% O(2); n = 5 each). Hypoxia increased ventilation and the number of Fos-IR nTS cells (21%, 13 ± 2; 12%, 58 ± 4; 10%, 166 ± 22; 8%, 186 ± 6). Fos expression after 10% O(2) was similar whether arterial pressure was allowed to decrease (-13 ± 1 mmHg) or was held constant. The percentage of PVN-projecting cells activated was intensity dependent, but contrary to our hypothesis, PVN-projecting nTS cells exhibiting Fos-IR were found at all hypoxic intensities. Notably, at all intensities of hypoxia, ∼75% of the activated PVN-projecting nTS neurons were catecholaminergic. Compared with RVLM-projecting cells, a greater percentage of PVN-projecting nTS cells was activated by 10% O(2). Data suggest that increasing hypoxic intensity activates nTS PVN-projecting cells, especially catecholaminergic, PVN-projecting neurons. The nTS to PVN catecholaminergic pathway may be critical even at lower levels of chemoreflex activation and more important to cardiorespiratory responses than previously considered.

Pregnancy and the endocrine regulation of the baroreceptor reflex.

The purpose of this review is to delineate the general features of endocrine regulation of the baroreceptor reflex, as well as specific contributions during pregnancy. In contrast to the programmed changes in baroreflex function that occur in situations initiated by central command (e.g., exercise or stress), the complex endocrine milieu often associated with physiological and pathophysiological states can influence the central baroreflex neuronal circuitry via multiple sites and mechanisms, thereby producing varied changes in baroreflex function. During pregnancy, baroreflex gain is markedly attenuated, and at least two hormonal mechanisms contribute, each at different brain sites: increased levels of the neurosteroid 3alpha-hydroxy-dihydroprogesterone (3alpha-OH-DHP), acting in the rostral ventrolateral medulla (RVLM), and reduced actions of insulin in the forebrain. 3alpha-OH-DHP appears to potentiate baroreflex-independent GABAergic inhibition of premotor neurons in the RVLM, which decreases the range of sympathetic nerve activity that can be elicited by changes in arterial pressure. In contrast, reductions in the levels or actions of insulin in the brain blunt baroreflex efferent responses to increments or decrements in arterial pressure. Although plasma levels of angiotensin II are increased in pregnancy, this is not responsible for the reduction in baroreflex gain, although it may contribute to the increased level of sympathetic nerve activity in this condition. How these different hormonal effects are integrated within the brain, as well as possible interactions with additional potential neuromodulators that influence baroreflex function during pregnancy and other physiological and pathophysiological states, remains to be clearly delineated.

SENSORY AFFERENT AND HYPOXIA-MEDIATED ACTIVATION OF NUCLEUS TRACTUS SOLITARIUS NEURONS THAT PROJECT TO THE ROSTRAL VENTROLATERAL MEDULLA

The nucleus tractus solitarius (nTS) of the brainstem receives sensory afferent inputs, processes that information, and sends projections to a variety of brain regions responsible for influencing autonomic and respiratory output. The nTS sends direct projections to the rostral ventrolateral medulla (RVLM), an area important for cardiorespiratory reflexes and homeostasis. Since the net reflex effect of nTS processing ultimately depends on the properties of output neurons, we determined the characteristics of these RVLM-projecting nTS neurons using electrophysiological and immunohistochemical techniques. RVLM-projecting nTS neurons were identified by retrograde tracers. Patch clamp analysis in the horizontal brainstem nTS slice demonstrated that RVLM-projecting nTS cells exhibit constant latency solitary tract evoked excitatory postsynaptic currents (EPSCs), suggesting they receive strong monosynaptic contacts from visceral afferents. Three distinct patterns of action potential firing, associated with different underlying potassium currents, were observed in RVLM-projecting cells. Following activation of the chemoreflex in conscious animals by 3 h of acute hypoxia, 11.2+/-1.9% of the RVLM-projecting nTS neurons were activated, as indicated by positive Fos-immunoreactivity. Very few RVLM-projecting nTS cells were catecholaminergic. Taken together, these data suggest that RVLM projecting nTS neurons receive strong monosynaptic inputs from sensory afferents and a subpopulation participates in the chemoreflex pathway.

Regulation of arterial pressure by the paraventricular nucleus in conscious rats: interactions among glutamate, GABA, and nitric oxide.

The paraventricular nucleus (PVN) of the hypothalamus is an important site for autonomic and neuroendocrine regulation. Experiments in anesthetized animals and in vitro indicate an interaction among gamma-aminobutyric acid (GABA), nitric oxide (NO), and glutamate in the PVN. The cardiovascular role of the PVN and interactions of these neurotransmitters in conscious animals have not been evaluated fully. In chronically instrumented conscious rats, mean arterial pressure (MAP) and heart rate (HR) responses to microinjections (100 nl) in the region of the PVN were tested. Bilateral blockade of ionotropic excitatory amino acid (EAA) receptors (kynurenic acid, Kyn) in the PVN produced small but significant decreases in MAP and HR. GABAA receptor blockade (bicuculline, Bic), and inhibition of NO synthase [(NOS), N-(G)-monomethyl-L-arginine, L-NMMA] each increased MAP and HR. The NO donor sodium nitroprusside (SNP) produced depressor responses that were attenuated by Bic. NOS inhibition potentiated both pressor responses to the selective EAA agonist, N-methyl-D-aspartic acid (NMDA), and depressor responses to Kyn. Increases in MAP and HR due to Bic were blunted by prior blockade of EAA receptors. Thus, pressor responses to GABA blockade require EAA receptors and GABA neurotransmission contributes to NO inhibition. Tonic excitatory effects of glutamate in the PVN are tonically attenuated by NO. These data demonstrate that, in the PVN of conscious rats, GABA, glutamate, and NO interact in a complex fashion to regulate arterial pressure and HR under normal conditions.

Fos expression in brain stem nuclei of pregnant rats after hydralazine-induced hypotension

Fos and dopamine beta-hydroxylase immunoreactivity were evaluated in the brain stems of 21-day pregnant and virgin female rats injected with either hydralazine (HDZ; 10 mg/kg iv) or vehicle. HDZ produced significant hypotension in both groups, although baseline blood pressure was lower in pregnant rats (96 +/- 2.5 mmHg) than in virgin female rats (121 +/- 2.8 mmHg). There were no differences in Fos immunoreactivity in the brain stems of pregnant and virgin female rats after vehicle treatment. HDZ-induced hypotension significantly increased Fos expression in both groups; however, the magnitude of the increases differed in the caudal ventrolateral medulla (CVL), the area postrema (AP), and the rostral ventrolateral medulla (RVL). Fos expression after HDZ in pregnant rats was augmented in noncatecholaminergic neurons of the CVL but was attenuated in the AP and in noncatecholaminergic neurons in the RVL. These results are consistent with differences in the sympathetic response to hypotension between pregnant and virgin female rats and indicate that the central response to hypotension may be different in pregnant rats.Fos and dopamine β-hydroxylase immunoreactivity were evaluated in the brain stems of 21-day pregnant and virgin female rats injected with either hydralazine (HDZ; 10 mg/kg iv) or vehicle. HDZ produced significant hypotension in both groups, although baseline blood pressure was lower in pregnant rats (96 ± 2.5 mmHg) than in virgin female rats (121 ± 2.8 mmHg). There were no differences in Fos immunoreactivity in the brain stems of pregnant and virgin female rats after vehicle treatment. HDZ-induced hypotension significantly increased Fos expression in both groups; however, the magnitude of the increases differed in the caudal ventrolateral medulla (CVL), the area postrema (AP), and the rostral ventrolateral medulla (RVL). Fos expression after HDZ in pregnant rats was augmented in noncatecholaminergic neurons of the CVL but was attenuated in the AP and in noncatecholaminergic neurons in the RVL. These results are consistent with differences in the sympathetic response to hypotension between pregnant and virgin female rats and indicate that the central response to hypotension may be different in pregnant rats.

Nitric oxide synthase activity and expression are decreased in the paraventricular nucleus of pregnant rats

Pregnancy is characterized by elevated heart rate and decreased total peripheral resistance and arterial blood pressure. Plasma volume is expanded and plasma osmolality is decreased, yet vasopressin secretion in pregnant animals, including humans, is no different than levels in the nonpregnant state. Although reflex compensatory sympathoexcitation is suppressed, baseline sympathetic nerve activity to the heart and vasculature is well maintained or slightly elevated in pregnancy. Clearly there are central nervous system (CNS) adaptations in systems for regulation of cardiovascular and body fluid homeostasis in pregnant animals. The paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus are important CNS sites for control of sympathetic nerve activity and vasopressin secretion. Nitric oxide (NO), an important neuromodulator in these hypothalamic nuclei, contributes to tonic inhibition of neurosecretory and pre-autonomic neurons. Alterations in NO within the PVN and SON could contribute to changes in regulation of vasopressin and sympathetic nerve activity in pregnancy. In the present study, nitric oxide synthase (NOS) activity (NADPH-diaphorase staining), neuronal NOS (nNOS) protein, and nNOS mRNA were assessed in nonpregnant estrus stage and near-term pregnant rats. nNOS mRNA, protein, and activity were greater in the PVN than in the SON. In the PVN only, pregnancy was associated with significant decreases in all three measurements for assessment of nNOS. Thus decreased NO production and relative disinhibition of the PVN may contribute to maintenance of baseline vasopressin secretion and baseline sympathetic nerve activity in the pregnant state.

Sex differences in baroreflex sensitivity, heart rate variability, and end organ damage in the TGR(mRen2)27 rat

The aim of this investigation was to evaluate sex differences in baroreflex and heart rate variability (HRV) dysfunction and indexes of end-organ damage in the TG(mRen2)27 (Ren2) rat, a model of renin overexpression and tissue renin-angiotensin-aldosterone system overactivation. Blood pressure (via telemetric monitoring), blood pressure variability [BPV; SD of systolic blood pressure (SBP)], spontaneous baroreflex sensitivity, HRV [HRV Triangular Index (HRV-TI), standard deviation of the average NN interval (SDNN), low and high frequency power (LF and HF, respectively), and Poincaré plot analysis (SD1, SD2)], and cardiovascular function (pressure-volume loop analysis and proteinuria) were evaluated in male and female 10-wk-old Ren2 and Sprague Dawley rats. The severity of hypertension was greater in Ren2 males (R2-M) than in Ren2 females (R2-F). Increased BPV, suppression of baroreflex gain, decreased HRV, and associated end-organ damage manifested as cardiac dysfunction, myocardial remodeling, elevated proteinuria, and tissue oxidative stress were more pronounced in R2-M compared with R2-F. During the dark cycle, HRV-TI and SDNN were negatively correlated with SBP within R2-M and positively correlated within R2-F; within R2-M, these indexes were also negatively correlated with end-organ damage [left ventricular hypertrophy (LVH)]. Furthermore, within R2-M only, LVH was strongly correlated with indexes of HRV representing predominantly vagal (HF, SD1), but not sympathetic (LF, SD2), variability. These data demonstrated relative protection in females from autonomic dysfunction and end-organ damage associated with elevated blood pressure in the Ren2 model of hypertension.

RVLM GLYCINE RECEPTORS MEDIATE GABAA AND GABAB INDEPENDENT SYMPATHOINHIBITION FROM CVLM IN RATS.

The caudal ventrolateral medulla (CVLM) provides tonic inhibitory and also excitatory inputs to the rostral ventrolateral medulla (RVLM). These experiments evaluated the role of RVLM gamma-amino butyric acid (GABA) receptor subtypes and glycine receptors in mediating CVLM sympathoinhibition. In Inactin anesthetized female rats, the CVLM and RVLM were functionally defined by pressor and depressor responses to microinjected GABA (500 pmol, 50 nl). Although reduced, pressor and sympathoexcitatory responses due to inhibition of the CVLM with GABA persisted following ipsilateral RVLM GABA(A) receptor blockade (bicuculline, BIC, 400 pmol, 100 nl; n=12) in rats with contralateral nucleus tractus solitarius (NTS) lesion. In the presence of either ipsilateral (+contralateral NTS lesion; n=8) or bilateral (n=6) GABA(A) and GABA(B) receptor blockade of the RVLM (400 pmol BIC+400 pmol CGP35348, 100 nl), inhibition of the CVLM still increased MAP and renal sympathetic nerve activity (RSNA). Thus neither GABA(B) receptors nor a contralateral CVLM to RVLM GABAergic pathway explains residual responses to CVLM blockade. The addition of strychnine (300 pmol, 100 nl) to the RVLM eliminated responses to CVLM inhibition, suggesting that a GABA(A) and GABA(B) independent sympathoinhibitory influence from CVLM to RVLM is mediated by glycine receptors. Decreases in MAP and RSNA due to activation of the CVLM with glutamate (500 pmol, 50 nl) were reversed to increases in the presence of RVLM GABA(A) receptor blockade (n=7). Thus, a sympathoexcitatory pathway from the CVLM can be activated in the presence of RVLM GABA receptor blockade, but sympathoinhibitory influences from the CVLM predominate.

EXPRESSION OF GROUP I METABOTROPIC GLUTAMATE RECEPTORS ON PHENOTYPICALLY DIFFERENT CELLS WITHIN THE NUCLEUS OF THE SOLITARY TRACT IN THE RAT

Group I metabotropic glutamate receptors (mGluRs) are G-coupled receptors that modulate synaptic activity. Previous studies have shown that Group I mGluRs are present in the nucleus of the solitary tract (NTS), in which many visceral afferents terminate. Microinjection of selective Group I mGluR agonists into the NTS results in a depressor response and decrease in sympathetic nerve activity. There is, however, little evidence detailing which phenotypes of neurons within the NTS express Group I mGluRs. In brainstem slices, we performed immunohistochemical localization of Group I mGluRs and either glutamic acid decarboxylase 67 kDa isoform (GAD67), neuronal nitric oxide synthase (nNOS) or tyrosine hydroxylase (TH). Fluoro-Gold (FG, 2%; 15 nl) was microinjected in the caudal ventrolateral medulla (CVLM) of the rat to retrogradely label NTS neurons that project to CVLM. Group I mGluRs were distributed throughout the rostral-caudal extent of the NTS and were found within most NTS subregions. The relative percentages of Group I mGluR expressing neurons colabeled with the different markers were FG (6.9+/-0.7) nNOS (5.6+/-0.9), TH (3.9+/-1.0), and GAD67 (3.1+/-1.4). The percentage of FG containing cells colabeled with Group I mGluR (13.6+/-2.0) was greater than the percent colabeled with GAD67 (3.1+/-0.5), nNOS (4.7+/-0.5), and TH (0.1+/-0.08). Cells triple labeled for FG, nNOS, and Group I mGluRs were identified in the NTS. Thus, these data provide an anatomical substrate by which Group I mGluRs could modulate activity of CVLM projecting neurons in the NTS.