Eugene V. Golanov

Inhibition of Nitric Oxide Synthesis Increases Focal Ischemic Infarction in Rat

We investigated whether inhibition of nitric oxide (NO) biosynthesis with N-ω-nitro-l-arginine (NNA), a competitive inhibitor of NO synthase (NOS), would modify the volume of the focal ischemic infarction produced by occlusion of the middle cerebral artery (MCA) in spontaneously hypertensive rats. NNA was infused for 1 h (2.4 mg/kg/h) immediately following occlusion of the MCA. NNA increased lesion volume 24 h later by 32% over controls (150.8 ± 16.6 to 199.2 ± 17.4 mm3; p < 0.001, n = 6). This effect was antagonized by co-infusion of l- but not d-arginine. The antihypertensive rilmenidine (0.75 mg/kg) reduced the lesion by 27% (p < 0.05, n = 4). Changes in lesion size were confined to the penumbra. NNA increased arterial pressure (AP) (118 ± 8.9 to 149 ± 16.0 mm Hg; p < 0.01, n = 3) but did not change regional CBF. However, elevation of AP did not change the lesion volume or distribution. We conclude that inhibition of the constitutive form of NOS in vivo increases the volume of focal ischemic infarction as a consequence of reduced NO biosynthesis. The absence of NO availability may extend lesion formation by inhibition of reactive hyperemia, platelet disaggregation, and/or release of neuroprotective neuromodulators in the penumbra, which may counteract and override any of its neurotoxic actions.

Biology of Vascular Malformations of the Brain

Background and Purpose— This review discusses recent research on the genetic, molecular, cellular, and developmental mechanisms underlying the etiology of vascular malformations of the brain (VMBs), including cerebral cavernous malformation, sporadic brain arteriovenous malformation, and the arteriovenous malformations of hereditary hemorrhagic telangiectasia. Summary of Review— The identification of gene mutations and genetic risk factors associated with cerebral cavernous malformation, hereditary hemorrhagic telangiectasia, and sporadic arteriovenous malformation has enabled the development of animal models for these diseases and provided new insights into their etiology. All of the genes associated with VMBs to date have known or plausible roles in angiogenesis and vascular remodeling. Recent work suggests that the angiogenic process most severely disrupted by VMB gene mutation is that of vascular stabilization, the process whereby vascular endothelial cells form capillary tubes, strengthen their intercellular junctions, and recruit smooth muscle cells to the vessel wall. In addition, there is now good evidence that in some cases, cerebral cavernous malformation lesion formation involves a genetic 2-hit mechanism in which a germline mutation in one copy of a cerebral cavernous malformation gene is followed by a somatic mutation in the other copy. There is also increasing evidence that environmental second hits can produce lesions when there is a mutation to a single allele of a VMB gene. Conclusions— Recent findings begin to explain how mutations in VMB genes render vessels vulnerable to rupture when challenged with other inauspicious genetic or environmental factors and have suggested candidate therapeutics. Understanding of the cellular mechanisms of VMB formation and progression in humans has lagged behind that in animal models. New knowledge of lesion biology will spur new translational work. Several well-established clinical and genetic database efforts are already in place, and further progress will be facilitated by collaborative expansion and standardization of these.

Adrenergic and non-adrenergic spinal projections of a cardiovascular-active pressor area of medulla oblongata: quantitative topographic analysis

A cardiovascular-active pressor area of medullary reticular formation was defined by mapping changes in arterial blood pressure produced by microinjections of the neuroexcitatory amino acid, L-Glutamate (L-Glu). Sites where L-Glu provoked pressor responses larger than 10 mmHg were localized to a rostral longitudinal cell column of the nucleus reticularis rostroventrolateralis (n.RVL) extending 450 microns posteriorly to the facial nucleus. Spinal projections from the ventrolateral medulla were studied with a dual retrograde transport-immunocytochemical method. A striking correspondence was observed between the ventrolateral pressor area (VLPA) of n.RVL and rostrocaudal distribution of a circumscribed population of thoracic reticulospinal neurons containing tyrosine hydroxylase (TH)- or phenylethanolamine N-methyltransferase (PNMT)_immunoreactivity. Quantitative analysis revealed that 72% of the total number of retrogradely labeled neurons within the active area were immunocytochemically positive for TH; 28% of the reticulospinal projection cells were immunonegative. Deposits of L-Glu and dye through the same micropipettes verified a consistent correlation of vasopressor sites and the rostral subset of catecholaminergic neurons. Since comparable numbers of cell bodies in the VLPA contain TH and PNMT all are presumed to be adrenergic. At levels of n.RVL immediately adjacent to the VLPA commencing at a level 450 microns caudal to the facial nucleus, sites were unresponsive to Glu-stimulation or vasodepressor. At these levels, only non-adrenergic reticulospinal neurons project to cervical or thoracic spinal segments. We conclude that the VLPA is highly restricted to a narrow column of n.RVL < 0.5 mm in length and corresponds precisely with a population of predominantly adrenergic thoracic reticulospinal neurons that project exclusively to sympathoadrenal preganglionic motoneurons [cf 46]. These findings corroborate the idea that an adrenergic-spinal pathway may play a role in controlling sympathetic outflow.

Autonomic and Vasomotor Regulation

The cerebellum not only modulates the systemic circulation, but also profoundly influences cerebral blood flow (rCBF) and metabolism (rCGU), and initiates long-term protection of the brain from ischemia. Electrical stimulation of the rostral ventral pole of the fastigial nucleus (FN), elevates arterial pressure (AP), releases vasoactive hormones, elicits consummatory behavioral and other autonomic events and site specifically elevates rCBF independently of changes in rCGU. Cerebral vasodilation results from the antidromic excitation of axons of brain stem neurons which innervate cerebellum and, through their collaterals, neurons in the rostral ventrolateral reticular nucleus (RVL). RVL neurons initiate cerebral vasodilation over polysynaptic vasodilator pathways which engage a population of vasodilator neurons in the cerebral cortex. In contrast, intrinsic neurons of FN, when excited, elicit widespread reductions in rCGU and, secondarily, rCBF, along with sympathetic inhibition. Electrical stimulation of FN can reduce the volume of a focal cerebral infarction produced by occlusion of the middle cerebral artery by 50%. This central neurogenic neuroprotection is long lasting (weeks) and is not due to changes in rCBF or rCGU. Rather, it appears to reflect alterations in neuronal excitability and/or downregulation of inflammatory responses in cerebral vessels. The FN, therefore, appears to be involved in widespread autonomic, metabolic, and behavioral control, independent of motor control. The findings imply that the FN receives inputs from neurons, probably widely represented in the central autonomic core, which may provide continuing information processing of autonomic and behavioral states. The cerebellum may also widely modulate the state of cortical reactivity to ischemia, hypoxia, and possibly other neurodegenerative events.

Central neurogenic neuroprotection: Central neural systems that protect the brain from hypoxia and ischemia

The brain can protect itself from ischemia and/or hypoxia by two distinct mechanisms which probably involve two separate systems of neurons in the CNS. One, which mediates a reflexive neurogenic neuroprotection, emanates from oxygen-sensitive sympathoexcitatory reticulospinal neurons of the RVLM. These cells, excited within seconds by reduction in blood flow or oxygen, initiate the systemic vascular components of the oxygen conserving (diving) reflex. They profoundly increase rCBF without changing rCGU and, hence, rapidly and efficiently provide the brain with oxygen. Upon cessation of the stimulus the systemic and cerebrovascular adjustments return to normal. The system mediating reflex protection projects via as-yet-undefined projections from RVLM to upper brainstem and/or thalamus to engage a small population of neurons in the cortex which appear to be dedicated to transducing a neuronal signal into vasodilation. It also appears to relay the central neurogenic vasodilation elicited from other brain regions, including excitation of axons innervating the FN. This mode of protection would be initiated under conditions of global ischemia and/or hypoxemia because the signal is detected by medullary neurons. The second neuroprotective system is represented in intrinsic neurons of the cerebellar FN and mediates a conditioned central neurogenic neuroprotection. The response can be initiated by excitation of intrinsic neurons of the FN and does not appear dependent upon RVLM. The pathways and transmitters that mediate the effect are unknown. The neuroprotection afforded by this network is long-lasting, persisting for almost two weeks, and is associated with reduced excitability of cortical neurons and reduced immunoreactivity of cerebral microvessels. This mode of neuroprotection, moreover, is not restricted to focal ischemia, as we have demonstrated that it also protects the brain against global ischemia and excitotoxic cell death. That the brain may have neuronal systems dedicated to protecting itself from injury, at first appearing to be a novel concept, is, upon reflection, not surprising since the brain is not injured in naturalistic behaviors characterized by very low levels of rCBF, diving and hibernation. An understanding of the pathways, transmitters, and molecules engaged in such protection may provide new insights into novel therapies for a range of disorders characterized by neuronal death.

Nitric oxide and prostanoids participate in cerebral vasodilation elicited by electrical stimulation of the rostral ventrolateral medulla.

We investigated, using laser-Doppler flowmetry, whether nitric oxide (NO)- and/or indomethacin (IND)-sensitive mechanisms mediate the elevations of regional cerebral blood flow (rCBF) elicited by electrical stimulation of the rostral ventrolateral medulla (RVL) in the anesthetized spinalized rat. Stimulation of the RVL for 10 s caused increased rCBF in the frontal cortex by 31% (n = 46), peaking at 22 s and persisting for up to 8 min. Intravenous l-nitro-NG-arginine (NNA) dose dependently and reversibly increased arterial pressure and reduced basal and evoked rCBF to 74 and 54% of the control, respectively (p < 0.05; n = 7). Superfused over the cortex, NNA dose dependently reduced only the evoked elevations of rCBF, to 39% of the control (p < 0.05; n = 6). Intravenous IND decreased the basal rCBF dose dependently and decreased the elevations evoked from the RVL by 38% (p < 0.05), but IND was without effect when superfused. Combined, the effects of intravenous NNA and IND summated, reducing rCBF by 70%. However, when NNA and IND were superfused together, the inhibition of the evoked vasodilation was comparable to that elicited by NNA alone. We conclude that the elevation in rCBF elicited from the RVL is partially mediated by (a) NO synthesized locally in the cortex in response to an afferent neural signal and (b) an IND-sensitive mechanism, probably a product of cyclooxygenase, located in larger cerebral arteries, in response to a retrograde vascular signal resulting from increased blood flow within the brain.

Cerebellar stimulation reduces inducible nitric oxide synthase expression and protects brain from ischemia

A focal infarction produced by occlusion of the middle cerebral artery (MCAO) in spontaneously hypertensive rats induced expression of inducible nitric oxide synthase (iNOS) mRNA, measured by competitive reverse transcription-polymerase chain reaction. The mRNA appeared simultaneously in the ischemic core and penumbra at 8 h, peaked between 14 and 24 h, and disappeared by 48 h. At 24 h, inducible nitric oxide synthase (iNOS)-like immunoreactivity was present in the endothelium of cerebral microvessels and in scattered cells, probably representing leukocytes or activated microglia. Electrical stimulation of the cerebellar fastigial nucleus (FN) for 1 h, 48 h before MCAO, reduced infarct volumes by 45% by decreasing cellular death in the ischemic penumbra. It also reduced by >90% the expression of iNOS mRNA and protein in the penumbra, but not core, and decreased by 44% the iNOS enzyme activity. We conclude that excitation of neuronal networks represented in the cerebellum elicits a conditioned central neurogenic neuroprotection associated with the downregulation of iNOS mRNA and protein. This neuroimmune interaction may, by blocking the expression of iNOS, contribute to neuroprotection.A focal infarction produced by occlusion of the middle cerebral artery (MCAO) in spontaneously hypertensive rats induced expression of inducible nitric oxide synthase (iNOS) mRNA, measured by competitive reverse transcription-polymerase chain reaction. The mRNA appeared simultaneously in the ischemic core and penumbra at 8 h, peaked between 14 and 24 h, and disappeared by 48 h. At 24 h, inducible nitric oxide synthase (iNOS)-like immunoreactivity was present in the endothelium of cerebral microvessels and in scattered cells, probably representing leukocytes or activated microglia. Electrical stimulation of the cerebellar fastigial nucleus (FN) for 1 h, 48 h before MCAO, reduced infarct volumes by 45% by decreasing cellular death in the ischemic penumbra. It also reduced by >90% the expression of iNOS mRNA and protein in the penumbra, but not core, and decreased by 44% the iNOS enzyme activity. We conclude that excitation of neuronal networks represented in the cerebellum elicits a conditioned central neurogenic neuroprotection associated with the downregulation of iNOS mRNA and protein. This neuroimmune interaction may, by blocking the expression of iNOS, contribute to neuroprotection.

Contribution of oxygen‐sensitive neurons of the rostral ventrolateral medulla to hypoxic cerebral vasodilatation in the rat.

1. We sought to determine whether hypoxic stimulation of neurons of the rostral ventrolateral reticular nucleus (RVL) would elevate regional cerebral blood flow (rCBF) in anaesthetized paralysed rats. 2. Microinjection of sodium cyanide (NaCN; 150‐450 pmol) into the RVL rapidly (within 1‐2 s), transiently, dose‐dependently and site‐specifically elevated rCBF1 measured by laser Doppler flowmetry, by 61.3 +/‐ 22.1% (P < 0.01), increased arterial pressure (AP; +30 +/‐ 8 mmHg; P < 0.01)1 and triggered a synchronized 6 Hz rhythm of EEG activity. 3. Following cervical spinal cord transection, NaCN and also dinitrophenol (DNP) significantly (P < 0.05) elevated rCBF and synchronized the EEG but did not elevate AP; the response to NaCN was attenuated by hyperoxia and deepening of anaesthesia. 4. Electrical stimulation of NaCN‐sensitive sites in the RVL in spinalized rats increased rCBF measured autoradiographically with 14C iodoantipyrine (Kety method) in the mid‐line thalamus (by 182.3 +/‐ 17.2%; P < 0.05) and cerebral cortex (by 172.6 +/‐ 15.6%; P < 0.05) regions, respectively, directly or indirectly innervated by RVL neurons, and in the remainder of the brain. In contrast regional cerebral glucose utilization (rCGU), measured autoradiographically with 14C‐2‐deoxyglucose (Sokoloff method), was increased in proportion to rCBF in the mid‐line thalamus (165.6 +/‐ 17.8%, P < 0.05) but was unchanged in the cortex. 5. Bilateral electrolytic lesions of NaCN sensitive sites of RVL, while not altering resting rCBF or the elevation elicited by hypercarbia (arterial CO2 pressure, Pa,CO2, approximately 69 mmHg), reduced the vasodilatation elicited by normocapnic hypoxaemia (arterial O2 pressure, Pa,O2, approximately 27 mmHg) by 67% (P < 0.01) and flattened the slope of the Pa,O2‐rCBF response curve. 6. We conclude that the elevation of rCBF produced in the cerebral cortex by hypoxaemia is in large measure neurogenic, mediated trans‐synaptically over intrinsic neuronal pathways, and initiated by excitation of oxygen sensitive neurons in the RVL.

Trigeminal Cardiac Reflex: New Thinking Model About the Definition Based on a Literature Review

AbstractTrigeminocardiac reflex (TCR) is a brainstem reflex that manifests as sudden onset of hemodynamic perturbation in blood pressure (MABP) and heart rate (HR), as apnea and as gastric hypermotility during stimulation of any branches of the trigeminal nerve. The molecular and clinical knowledge about the TCR is in a constant growth since 1999, what implies a current need of a review about its definition in this changing context.Relevant literature was identified through searching in PubMed (MEDLINE) and Google scholar database for the terms TCR, oculocardiac reflex, diving reflex, vasovagale response.The definition of the TCR varies in clinical as well as in research studies. The main difference applies the required change of MABP and sometimes also HR, which most varies between 10% and 20%. Due to this definition problem, we defined, related to actual literature, 2 major (plausibility, reversibility) and 2 minor criteria (repetition, prevention) for a more proper identification of the TCR in a clinical or research setting. Latest research implies that there is a need for a more extended classification with 2 additional subgroups, considering also the diving reflex and the brainstem reflex.In this review, we highlighted criteria for proper definition and classification of the TCR in the light of increased knowledge and present a thinking model to overcome this complexity. Further we separately discussed the role of HR and MABP and their variation in this context. As another subtopic we gave attention to is the chronic TCR; a variant that is rarely seen in clinical medicine.

Brief electrical stimulation of cerebellar fastigial nucleus conditions long-lasting salvage from focal cerebral ischemia: conditioned central neurogenic neuroprotection

The cerebellar fastigial nucleus (FN) was electrically stimulated for 1 h in anesthetized rats and the middle cerebral artery occluded at various times thereafter. Stimulation of the FN but not dentate nucleus reduced the volume of the focal infarction to 50%. Protection persisted for 10 but disappeared by 30 d. Intrinsic neuronal pathways which function to condition central neurogenic neuroprotection can protect the brain from ischemic injury by processes independent of cerebral blood flow.