Michal Horowitz

Fractionation of oxygen isotopes between mammalian bone-phosphate and environmental drinking water

The δ18O of mammalian bone-phosphate varies linearly with δ18O of environmental water, but is not in isotopic equilibrium with that water. This situation is explained by a model of δ18O in body water in which the important fluxes of exchangeable oxygen through the body are taken into account. Fractionation of oxygen isotopes between body and environmental drinking water is dependent on the rates of drinking and respiration. Isotopic fractionation can be estimated from physiological data and the estimates correlate very well with observed fractionation. Species whose water consumption is large relatively to its energy expenditure is sensitive to isotopic ratio changes in environmental water.

Oxidative Stress in Closed-Head Injury: Brain Antioxidant Capacity as an Indicator of Functional Outcome

It has been suggested that reactive oxygen species (ROS) play a role in the pathophysiology of brain damage. A number of therapeutic approaches, based on scavenging these radicals, have been attempted both in experimental models and in the clinical setting. In an experimental rat and mouse model of closed-head injury (CHI), we have studied the total tissue nonenzymatic antioxidant capacity to combat ROS. A major mechanism for neutralizing ROS uses endogenous low-molecular weight antioxidants (LMWA). This review deals with the source and nature of ROS in the brain, along with the endogenous defense mechanisms that fight ROS. Special emphasis is placed on LMWA such as ascorbate, urate, tocopherol, lipoic acid, and histidine-related compounds. A novel electrochemical method, using cyclic voltammetry for the determination of total tissue LMWA, is described. The temporal changes in brain LMWA after CHI, as part of the response of the tissue to high ROS levels, and the correlation between the ability of the brain to elevate LMWA and clinical outcome are addressed. We relate to the beneficial effects observed in heat-acclimated rats and the detrimental effects of injury found in apolipoprotein E-deficient mice. Finally, we summarize the effects of cerebroprotective pharmacological agents including the iron chelator desferal, superoxide dismutase, a stable radical from the nitroxide family, and HU-211, a nonpsychotoropic cannabinoid with antioxidant properties. We conclude that ROS play a key role in the pathophysiology of brain injury, and that their neutralization by endogenous or exogenous antioxidants has a protective effect. It is suggested, therefore, that the brain responds to ROS by increasing LMWA, and that the degree of this response is correlated with clinical recovery. The greater the response, the more favorable the outcome.

From molecular and cellular to integrative heat defense during exposure to chronic heat

Heat acclimation induces adaptive changes that improve the ability to cope with extreme environmental heat. Acclimatory homeostasis is manifested by an expanded dynamic thermoregulatory span (TRS), reflected in the intact organism by a lower temperature threshold (T(sh)) for heat dissipation, and delayed T(sh) for thermal injury. This principle shares common adaptive features with each of the thermoregulatory effectors. In the splanchnic circulation, e.g. the TRS of the thermally induced vasomotor response increases due to greater cardiac output distribution to the splanchnic vasculature, thereby increasing circulatory reserves and delaying thermal injury. During short-term heat acclimation (STHA), accelerated autonomic excitability plays a major role in the control of body temperature. Acclimatory homeostasis, however, is achieved only following long-term heat acclimation (LTHA), and is characterized by increased thermal effector efficiency, namely [effector organ output/autonomic signal] ratio >1. Two acclimatory responses, derived from our data on the acclimating rat model, are discussed: (1) acclimation of the cholinergic-muscarinic signaling for water secretion in the submaxillary gland; and (2) acclimatory mechanisms for increased contractile efficiency in the heart. Our data indicate that increased efficiency upon LTHA develops by reprogramming of gene expression. A reduced thyroid hormone level is responsible for some of the molecular adaptive cascades. Delayed thermal injury observed upon acclimation is due to enhanced cytoprotective mechanisms of which the inducible heat shock protein (HSP) 72 kDa plays a major role. Our data indicate that heat acclimation predisposes the HSP molecular machinery to respond faster and increases the constitutive level of the protein. STHA is the time-window during which most LTHA adaptations are switched on.

Heat acclimation increases the basal HSP72 level and alters its production dynamics during heat stress

It has been previously shown that heat acclimation leads to an elevated basal level of 72-kDa heat shock protein (HSP72). Augmented expression of HSP72 is considered as a cytoprotective response. This led us to hypothesize that alterations in the heat shock protein (HSP) defense pathway are an integral part of the heat acclimation repertoire. To investigate this, we studied the temporal profile of basal HSP expression upon acclimation and the dynamics of their accumulation subsequent to acute heat stress (HS). In parallel, HSP72 mRNA level before and after HS was measured. For comparison, HSC mRNA [the constitutive member of 70-kDa HSP (HSP70) family] was measured in similar conditions. Heat acclimation was attained by continuous exposure of rats to 34°C for 0, 1, 2, and 30 days. HS was attained by exposure to 41 or 43°C for 2 h. Thermoregulatory capacity of the rats was defined by rectal temperature, heating rate, and the cumulative heat strain invoked during HS. HSP72 and HSP70 gene transcripts were measured in the left ventricle of the heart by means of Western immunoblotting and semiquantitative RT-PCR, respectively. The resultant acclimatory change comprised a higher resting level of the encoded 72-kDa protein (Δ175%, P < 0.0001). After HS, peak HSP72 mRNA level was attained, 40 and 20 min post-HS at 41 and 43°C, respectively, vs. 60 and 40 min in the nonacclimated group. The subsequent HSP synthesis, however, was dependent on the severity of the cumulative heat strain. At the initial phase of heat acclimation, augmented HSP72 transcription unaccompanied by HSP synthesis was observed. It is concluded that upon heat acclimation, the HSP defense pathway is predisposed to a faster response. At the initial phases of heat acclimation, inability to elevate the HSP cytosolic level rules out their direct cytoprotective role.

Carbachol, an acetylcholine receptor agonist, enhances production in rat aorta of 2-arachidonoyl glycerol, a hypotensive endocannabinoid

The production of 2-arachidonoyl glycerol, an endogenous cannabinoid, is enhanced in normal, but not in endothelium-denuded rat aorta on stimulation with carbachol, an acetylcholine receptor agonist. 2-Arachidonoyl glycerol potently reduces blood pressure in rats and may represent an endothelium-derived hypotensive factor.

Changes of Biological Reducing Activity in Rat Brain following Closed Head Injury: A Cyclic Voltammetry Study in Normal and Heat-Acclimated Rats

Reactive oxygen species (ROS) are normally generated in the brain during metabolism, and their production is enhanced by various insults. Low molecular weight antioxidants (LMWA) are one of the defense mechanisms of the living cell against ROS. The reducing capacity of brain tissue (total LMWA) was measured by cyclic voltammetry (CV), which records biological oxidation potential specific to the type of scavenger(s) present and anodic current intensity (Ia), which depends on scavenger concentration. In the present study, the reducing capacity of rat brain following closed head injury (CHI) was measured. In addition, CV of heat-acclimated traumatized rats was used to correlate endogenous cerebroprotection after CHI with LMWA activity. Sham-injured rat brains displayed two anodic potentials: at 350 ± 50 mV (Ia = 0.75 ± 0.06 μA/mg protein) and at 750 ± 50 mV (Ia = 1.00 ± 0.05 μA/mg protein). Following CHI, the anodic waves appeared at the same potentials as in the sham animals. However, within 5 min of CHI, the total reducing capacity was transiently decreased by 40% (p < 0.01). A second dip was detected at 24 h (60%, p < 0.005). By 48 h and at 7 days, the Ia levels normalized. The acclimated rats displayed anodic potentials identical to those of normothermic rats. However, the Ia of both potentials was lower (60% of control, p < 0.001). The Ia profile after CHI was the direct opposite of the normothermic Ia profile: no immediate decrease of Ia and an increase from 4 h and up to 7 days (40–50%, p < 0.001). We suggest that the lowered levels of LMWA in the post-CHI period reflect their consumption due to overproduction of free radicals. The augmented concentration of LMWA found in the brain of the heat-acclimated rats suggests that these rats are better able to cope with these harmful radicals, resulting in a more favorable outcome following CHI.

Differential stimulation of c-fos expression in hypothalamic nuclei of the rat brain during short-term heat acclimation and mild dehydration

Activation of central nervous structures involved in the perception and integration of thermo- and osmoregulatory signals was investigated in the Sabra rat. Male rats were either non-treated (C-E), water-deprived for 24 h (C-D), short-term acclimated to 34 degrees C for two days (STHA-E) or subjected to both stimuli (STHA-D). Immunoreactivity for c-Fos protein (Fos-IR) as marker for neuronal activation was quantified in (extra-)hypothalamic structures: organum vasculosum laminae terminalis (OVLT); subfornical organ (SFO); medial (MPA), ventromedial preoptic (VMPO) and lateral hypothalamic (LHA) areas; median preoptic (MnPO), magnocellular supraoptic (SON) and paraventricular (mPVN) nuclei; limbic lateral septal (LS) and thalamic paraventricular (PV) nuclei. Compared to C-E rats, dehydration markedly increased Fos-IR exclusively in neurons of the OVLT, SFO and MnPO known to be involved in osmoreception, in the mPVN and SON, and to a minor extent in the VMPO. The VMPO, MPA, LHA and LS-important (extra-)hypothalamic sites for the perception and integration within the thermoregulatory control circuit-exhibited intense elevation of Fos-IR upon short-term heat acclimation. Of all (extra-)hypothalamic structures involved in central osmoregulation, only the MnPO revealed heat-induced Fos-IR in numerous cells located preferentially in its rostral component. Thus, the MnPO proved to be activated during both thermal and osmotic stimulations applied separately. Subjected to the combined stress (STHA-D), most brain structures investigated showed striking Fos-IR due to thermally enhanced osmotic stimulation, with additive effects demonstrated in the MnPO. The data support differential central activation of c-fos expression due to thermal or osmotic stimulations, with the MnPO acting as putative integrative center for both autonomic control circuits.

Acclimatization of rats to moderate heat: Body water distribution and adaptability of the submaxillary salivary gland

SummaryChanges in plasma and extracellular fluid volumes were studied in rats during exposure to 35° C for 28 days. In addition the adaptability of the submaxillary salivary gland to these conditions was studied by measuring its weight, plasma and extracellular volumes during the acclimatization period. Major changes in parameters studied occurred during the first 10 days of acclimatization. Total plasma volume on day 10 was less than in controls (P=0.02), but from then on returned to normal values. Extracellular fluid volume was expanded for most of the period. An enlargement of the submaxillary salivary gland was present during the whole period, but maximal enlargement was observed on day 10 (42%). On day 28 the gland was 14% larger. No changes in plasma and extracellular volumes of the gland were observed. It appears that due to redistribution of the blood pool at the beginning of heat exposure, plasma and extracellular volumes remain constant.

Heat Acclimation Increases Hypoxia-Inducible Factor 1α and Erythropoietin Receptor Expression: Implication for Neuroprotection after Closed Head Injury in Mice

Experimental evidence indicates that long-term exposure to moderately high ambient temperature (heat acclimation, HA) mediates cross-tolerance to various types of subsequently applied stress. The transcriptional activator hypoxia-inducible factor 1 (HIF-1) has been implicated in playing a critical role in HA. It also regulates the expression of Erythropoietin (Epo), whose neuroprotective effects have been shown in a variety of brain injuries. The aim of the present study was to examine whether HA exerts a beneficial effect on the outcome of closed head injury (CHI) in mice and to explore the possible involvement of HIF-1 and Epo in this process. Heat acclimated mice and matched normothermic controls were subjected to CHI or sham surgery. Postinjury motor and cognitive parameters of acclimated mice were compared with those of controls. Mice were killed at various time points after injury or sham surgery and brain levels of HIF-1α, the inducible subunit of HIF-1, Epo, and the specific erythropoietin receptor (EpoR) were analyzed by Western immunoblotting. Motor and cognitive functions of acclimated mice were significantly better than those of controls. Heat acclimation was found to induce a significant increase in expression of nuclear HIF-1α and EpoR. The EpoR/Epo ratio was also significantly higher in acclimated mice as compared with controls. Nuclear HIF-1α and EpoR were higher in the acclimated group at 4 h after injury as well. The improved outcome of acclimated mice taken together with the basal and postinjury upregulation of the examined proteins suggests the involvement of this pathway in HA-induced neuroprotection.

Thermoregulatory activity in the rat: Effects of hypohydration, hypovolemia and hypertonicity and their interaction with short-term heat acclimation

Hypothalamic temperature thresholds to heat-induced (40 degrees C ambient temperature) tail vasodilation (Vth) and salivation (Sth) as well as salivary flow rate and volume were studied in conscious rats, hypohydrated (24 hr water deprivation), hypovolemic (20% dextran sc), hypertonic (1M NaCL po), hypertonic and hypovolemic and heat-acclimated (5 days at 34 degrees C) before and after hypohydration. Sth was elevated in hypohydrated, hypovolemic, hypertonic and heat-acclimated hypohydrated rats concomitantly with a remarkable decrease in saliva volume, flow rate and heat tolerance. Heat acclimation alone resulted in a reduction in Vth, Sth, salivary flow and volume. Vth was not affected by hypohydration, but was elevated following hypovolemia and combined hypovolemia and hypertonicity. It is concluded that alterations in both plasma volume and osmolarity, which may occur during hypohydration, play a major role in the alteration in thermoregulatory responses during hypohydration. Heat acclimation does not improve tolerance during hypohydration. Thus, during hypohydration, the control of body fluids overrides thermoregulation.