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, 8, 18-27
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Remote Ischemic Conditioning Improves Outcome Independent of Anesthetic Effects Following Shockwave-Induced Traumatic Brain Injury

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Remote Ischemic Conditioning Improves Outcome Independent of Anesthetic Effects Following Shockwave-Induced Traumatic Brain Injury

Eugene Park et al. IBRO Rep.

Abstract

Traumatic brain injury due to primary blast exposure is a major cause of ongoing neurological and psychological impairment in soldiers and civilians. Animal and human evidence suggests that low-level blast exposure is capable of inducing white matter injury and behavioural deficits. There are currently no effective therapies to treat the underlying suspected pathophysiology of low-level primary blast or concussion. Remote ischemic conditioning (RIC) has been shown to have cardiac, renal and neuro-protective effects in response to brief cycles of ischemia. Here we examined the effects of RIC in two models of blast injury. We used a model of low-level primary blast in rats to evaluate the effects of RIC neurofilament expression. We subsequently used a model of traumatic brain injury in adult zebrafish using pulsed high intensity focused ultrasound (pHIFU) to evaluate the effects of RIC on behavioural outcome and apoptosis in a post-traumatic setting. In blast exposed rats, RIC pretreatment modulated NF200 expression suggesting an innate biological buffering effect. In zebrafish, behavioural deficits and apoptosis due to pHIFU-induced brain injury were reduced following administration of serum derived from RIC rats. The results in the zebrafish model demonstrate the humoral effects of RIC independent of anesthetic effects that were observed in the rat model of injury. Our results indicate that RIC is effective in improving outcome following modeled brain trauma in pre- and post-injury paradigms. The results suggest a potential role for innate biological systems in the protection against pathophysiological processes associated with impairment following shockwave induced trauma.

Keywords: Behavior; Blast injury; Concussion; Ischemic conditioning; Preconditioning; Primary blast; Traumatic brain injury; Zebrafish.

Figures

Fig. 1
Fig. 1
A schematic timeline of the study design. Rats were subject to either the RIC treatment (including anesthesia) or 40 min of anesthesia alone. Following a 1 h recovery, rats were subject to the primary blast injury. A subset of rats were sacrificed following recovery for serum collection for zebrafish studies. Behavioural testing was performed at 3, 6 and 9 days post-injury. Tissues for Western blot analysis were collected at 48 h post-injury. B) The schematic representation of the zebrafish brain indicates the sectioning plane used for TUNEL histology in adult zebrafish. The boxed areas in the adjacent diagram indicates the sampling regions from the telencephalon and mesencephalon used for TUNEL quantification analysis.
Fig. 2
Fig. 2
a & b) Expression of the heavy neurofilament, NF200, was examined in sham, blast and RIC + blast tissues. There was a notable reduction in intact NF200 expression in blast tissues and an increase in NF200 expression in RIC treated rats. Densitometry values between blast and blast + RIC tissues were significantly different (p = 0.004). c) Comparison of NF200 expression between treatment groups as well as the inclusion of anesthetic control tissues indicated a marked reduction in N200 in rats subject to anesthetic preconditioning alone. D) Comparison of the NF200 response to two levels of subclinical blast exposure indicates a reduction in overall NF200 expression in the higher blast (35 kPa) group, while the lower level blast injury (11.5 kPa) results in an increase in intact NF200 expression as well as evidence of lower molecular weight breakdown products.
Fig. 3
Fig. 3
a & b) Behavioural outcome in the open field test indicated a reduction in both rearings and overall exploration activity in blast rats. Relative to sham controls. Post-hoc p-values between sham and blast rats was *p = 0.032 and ** p = 0.040 between blast and isoflurane control rats. Similarly, although the rearing data suggest a protective effect mediated by isoflurane, values were not statistically significant. c) In the light-dark box test there was a significant reduction in time spent in the lit section in the blast group relative to sham rats (* p < 0.001) and isoflurane exposed rats (**p = 0.029) on day 6. On day 9 blast rats continue to have significantly lower exploration times than sham rats (* p = 0.003). Isoflurane preconditioning resulted in a significant increase in the time spent in the lit section following blast exposure compared to blast alone (** p < 0.001).
Fig. 4
Fig. 4
a) Injured fish administered anesthetic control serum (pHIFU + Iso) had reduced swim distance relative to controls whereas pHIFU + RIC serum treated fish had a significant improvement in total swim distance (*p < 0.001). b) Swim velocity was significantly reduced in pHIFU + Iso fish relative to controls and improved in fish treated with RIC serum (*p < 0.001). c) The data for meander outcomes suggested an increase in mean meander activity in pHIFU + Iso fish relative to control fish. RIC serum treatment reduced post-traumatic meander activity. d) Similarly, latency values for tank exploration were not significantly different among treatment groups but the data suggested an increase in latency times for exploration in pHIFU + Iso. RIC serum treated fish exhibited a reduction in the latency times relative to isoflurane control treated fish.
Fig. 5
Fig. 5
a) Summarized TUNEL counts from sampling regions of the adult zebrafish brain indicated a increased TUNEL labelling across the sampled regions suggesting that the pHIFU injury was not limited to a single focal area or structure. b) Representative TUNEL brain sections sampled from the cortex of the mesencephalon in sham, pHIFU + Iso and pHIFU + RIC serum treated fish. TUNEL positive cells indicated with arrowheads. c) Summarized total TUNEL count data indicates a significant increase in TUNEL positivity in pHIFU + Iso fish relative to sham and pHIFU + RIC serum treated fish (p < 0.001).

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