Personalized approach in brain protection by hypothermia: individual changes in non-pathological and ischemia-related glutamate transport in brain nerve terminals

Abstract

Background: Both deep and profound hypothermia are effectively applied in cardiac surgery of the aortic arch, when the reduction of cerebral circulation facilitates operations, and for the prevention of ischemic stroke consequences. Neurochemical discrimination of the effects of deep and profound hypothermia (27 and 17 °C, respectively) on non-pathological and pathological ischemia-related mechanisms of presynaptic glutamate transport with its potential contribution to predictive, preventive and personalized medicine (PPPM) was performed.

Methods: Experiments were conducted using nerve terminals isolated from rat cortex (synaptosomes). Glutamate transport in synaptosomes was analyzed using radiolabel l-[¹⁴C]glutamate. Diameter of synaptosomes was assessed by dynamic light scattering.

Results: Synaptosomal transporter-mediated uptake and tonic release of l-[¹⁴C]glutamate (oppositely directed processes, dynamic balance of which determines the physiological extracellular level of the neurotransmitter) decreased in a different range in deep/profound hypothermia. As a result, hypothermia-induced changes in extracellular l-[¹⁴C]glutamate are not evident (in one half of animals it increased, and in other it decreased). A progressive decrease from deep to profound hypothermia was shown for pathological mechanisms of presynaptic glutamate transport, that is, transporter-mediated l-[¹⁴C]glutamate release (*) stimulated by depolarization of the plasma membrane and (**) during dissipation of the proton gradient of synaptic vesicles by the protonophore FCCP.

Conclusions: Therefore, the direction of hypothermia-induced changes in extracellular glutamate is unpredictable in "healthy" nerve terminals and depends on hypothermia sensitivity of uptake vs. tonic release. In affected nerve terminals (e.g., in brain regions suffering from a reduction of blood circulation during cardiac surgery, and core and penumbra zones of the insult), pathological transporter-mediated glutamate release from nerve terminals decreases with progressive significance from deep to profound hypothermia, thereby underlying its potent neuroprotective action. So, alterations in extracellular glutamate during hypothermia can be unique for each patient. An extent of a decrease in pathological glutamate transporter reversal depends on the size of damaged brain zone in each incident. Therefore, test parameters and clinical criteria of neuromonitoring for the evaluation of individual hypothermia-induced effects should be developed and delivered in practice in PPPM.

Keywords: Brain nerve terminals; Deep and profound hypothermia; Glutamate; Glutamate transporter reversal; Individual hypothermia regime; Neuromonitoring; Permanent glutamate turnover; Uptake and tonic release.

Fig. 1

Dynamic light scattering histograms. Synaptosomal suspension (0.2 mg/ml) in a standard saline solution at 37 (a), 27 (b), and 17° C (c)

Fig. 2

Tonic release of l-[¹⁴C]glutamate from nerve terminals under conditions of deep and profound hypothermia. 37 (empty column), at 27 (grey column), and 17 °C (black column). l-[¹⁴C]glutamate release was measured as described in the “Methods” section. Total synaptosomal l-[¹⁴C]glutamate content was equal to 200,000 ± 15,000 cpm/mg protein. Data are means ± SEM of 30 independent experiments, each performed in triplicate. Data are compared by Student’s t test. *, P ≤ 0.001 as compared to synaptosomes at 37 °C; #, as compared to synaptosomes at 17 °C

Fig. 3

Time course of l-[¹⁴C]glutamate uptake by nerve terminals under conditions of deep and profound hypothermia. 37 (solid line), 27 (dash line), and 17 °C (dotted line). l-[¹⁴C]glutamate uptake was measured as described in the “Methods” section

Fig. 4

The extracellular level of l-[¹⁴C]glutamate in the synaptosomal suspension under conditions of deep and profound hypothermia. The level was measured at 14-min time point (see the “Methods” section) at 37 (empty column), 27 (grey column), and 17 °C (black column). Total synaptosomal l-[¹⁴C]glutamate content was equal to 200,000 ± 15,000 cpm/mg protein. Data are means ± SEM of 30 independent experiments, each performed in triplicate

Fig. 5

Two groups of animals with different changes in the extracellular level of l-[¹⁴C]glutamate in the synaptosomal suspension under conditions of deep and profound hypothermia. The experiments were subdivided in two groups with a tendency to decrease (a) and increase (b) in the value of extracellular l-[¹⁴C]glutamate in the synaptosomal suspension in hypothermia. The level was measured at 14-min time point (see the “Methods” section) at 37 (empty columns), 27 (grey columns), and 17 °C (black columns). Total synaptosomal l-[¹⁴C]glutamate content was equal to 200,000 ± 15,000 cpm/mg protein. Data are means ± SEM of 30 independent experiments, each performed in triplicate

Fig. 6

Stimulated by high KCl (35 mM) transporter-mediated release of l-[¹⁴C]glutamate from nerve terminals under conditions of deep and profound hypothermia. Release was measured at 37 (empty columns), 27 (grey columns), and 17 °C (black columns). l-[¹⁴C]glutamate release was measured as described in the “Methods” section, and l-[¹⁴C]glutamate radioactivity was determined at 6-min time point. Data are means ± SEM of five independent experiments, each performed in triplicate. *, P ≤ 0.05 as compared to the control

Fig. 7

Transporter-mediated release of l-[¹⁴C]glutamate from nerve terminals in the presence of the protonophore FCCP (1 μM) under conditions of deep and profound hypothermia. l-[¹⁴C]glutamate release was measured as described in the “Methods” section, and l-[¹⁴C]glutamate radioactivity was determined at 6-min time point. 37 (empty columns), 27 (grey columns), and 17 °C (black columns). Data are means ± SEM of four independent experiments, each performed in triplicate. *, P ≤ 0.05 as compared to control

Fig. 8

Roadmap of the study