Hypothermia protects human neurons

Abstract

Background and aims: Hypothermia provides neuroprotection after cardiac arrest, hypoxic-ischemic encephalopathy, and in animal models of ischemic stroke. However, as drug development for stroke has been beset by translational failure, we sought additional evidence that hypothermia protects human neurons against ischemic injury.

Methods: Human embryonic stem cells were cultured and differentiated to provide a source of neurons expressing β III tubulin, microtubule-associated protein 2, and the Neuronal Nuclei antigen. Oxygen deprivation, oxygen-glucose deprivation, and H2 O2 -induced oxidative stress were used to induce relevant injury.

Results: Hypothermia to 33°C protected these human neurons against H2 O2 -induced oxidative stress reducing lactate dehydrogenase release and Terminal deoxynucleotidyl transferase dUTP nick end labeling-staining by 53% (P ≤ 0·0001; 95% confidence interval 34·8-71·04) and 42% (P ≤ 0·0001; 95% confidence interval 27·5-56·6), respectively, after 24 h in culture. Hypothermia provided similar protection against oxygen-glucose deprivation (42%, P ≤ 0·001, 95% confidence interval 18·3-71·3 and 26%, P ≤ 0·001; 95% confidence interval 12·4-52·2, respectively) but provided no protection against oxygen deprivation alone. Protection (21%) persisted against H2 O2 -induced oxidative stress even when hypothermia was initiated six-hours after onset of injury (P ≤ 0·05; 95% confidence interval 0·57-43·1).

Conclusion: We conclude that hypothermia protects stem cell-derived human neurons against insults relevant to stroke over a clinically relevant time frame. Protection against H2 O2 -induced injury and combined oxygen and glucose deprivation but not against oxygen deprivation alone suggests an interaction in which protection benefits from reduction in available glucose under some but not all circumstances.

Keywords: brain; hypothermia; ischemic stroke; neuroprotection; stem cells; treatment.

Figure 1

β III tubulin positive human neurons after 11 days in culture with nuclei counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI).

Figure 2

The effects of hypothermia on H₂O₂-induced cell death. Hypothermia reduces LDH detected cell death by 45% at four-hours (a) and effectively abolished (92% reduction) the delayed injury (b). The net effect at 24 h was reduction of 53% (c). H₂O₂-induced oxidative stress increased TUNEL-detected apoptotic cell death 2.9-fold after 24 h and hypothermia prevents 42% of this death (d). Statistical significance detected at *P ≤ 0·05, **P ≤ 0·01, #P ≤ 0·0005, and ##P ≤ 0·0001. Data presented as mean ± SEM.

Figure 3

The effects of hypothermia on oxygen deprivation induced cell death. Hypothermia has no beneficial effect at four hours (a), 4–20 h (b), or in the combined 24 h LDH measurement of cell death (c) as well as on apoptotic cell death detected by TUNEL staining at 24 h (d). NS = not significant.

Figure 4

The effects of hypothermia on oxygen glucose deprivation (OGD)-induced cell death. Hypothermia reduced OGD-induced cell death at four hours by 37% (a). Hypothermia reduced the delayed injury that occurred between removal of OGD at four hours and completion of the experiment at 24 h by 80% (b). The net effect at 24 h was a reduction of LDH release of 42% (c). TUNEL staining for apoptosis at 24 h suggested that 22% of cell death occurred by this mechanism and that hypothermia prevented 26% of this death (d). *P ≤ 0·05, #P ≤ 0·0005, and ##P ≤ 0·0001. Data presented as mean ± SEM.

Figure 5

Time-dependent effects of hypothermia on H₂O₂ and OGD-induced cell death. Hypothermia reduces LDH-detected cell death induced with H₂O₂ by 52%, 43%, 34%, and 21% (a) and OGD-induced injury by 45%, 30%, 27%, and 4% when started at 0, one-, three-, and six-hours (b), respectively. *P ≤ 0·05, **P ≤ 0·01, #P ≤ 0·0005, and ##P ≤ 0·0001. Data presented as mean ± SEM.