TBI is acquired from an external force, possibly resulting in devastating effects to both the cerebrovasculature and neighboring neuronal cells.10 Disruption of the macroscopic components and microvasculature occurs with the initial primary injury, and often initiates a host of secondary processes that can worsen the impact of the primary injury. The cerebral microcirculation, the main transport and distribution system for oxygen to the cerebral tissue, is significantly compromised in the peri-contusional area after TBI + HS.35 Despite advances in the understanding of TBI + HS, existing resuscitation approaches inadequately modulate CBF via the microcirculation, and fail to restore normal microcirculatory perfusion leading to hypoxia after TBI + HS as well as may perpetuate reperfusion injury.7,36,37 Therefore, novel resuscitation strategies that can enhance cerebral microvascular perfusion and, thus, oxygen delivery to tissues in peri-contusional areas are highly desirable. In the present study, we demonstrate in a rat model of TBI + HS that low-frequency CBF oscillations (0.1 Hz) delivered after TBI + HS play a more significant role than improving non-oscillatory cerebral perfusion, by mitigating secondary brain injury, attenuating brain hypoxic injury and neuroinflammation, protecting cerebrovascular endothelium in peri-contusional brain tissue, enhancing neurological recovery, and improving survival rate. To our knowledge, this is the first study where the TNS-induced low-frequency oscillations in CBF have been applied as a therapeutic strategy in TBI + HS.
Spontaneous low-frequency oscillations (0.04–0.15 Hz) in CBF have been observed in individuals with delayed syncope after induced central hypovolemia and are thought to preserve cerebral perfusion.17–20 While the physiologic mechanisms that govern CBF oscillations remain unclear, studies have shown that oscillatory CBF modulates the release of vasoactive molecules important in maintaining cerebral perfusion via flow-mediated regulatory mechanisms.38 Specifically, pulsatile blood flow can increase shear stress on the vessel endothelium which stimulates the release of nitric oxide and inhibits endothelin production, thus increasing oxygen delivery to brain tissue.21 Furthermore, these oscillatory patterns represent an “on-off” feedback mechanism that serves to maintain tissue perfusion in the setting of oxygen deprivation with computational models suggesting that low frequency oscillatory flow is beneficial to tissue oxygenation.15,16 It might be that brief, rhythmic increases in CBF during the oscillatory stimulus (Fig. 1B, Fig. 1C) provide a pump-like effect leading to improved tissue perfusion via temporary increases in the pressure gradient down the vascular tree. The low-frequency CBF oscillations supply higher amplitudes of localized oxygen delivery relative to oscillations at a higher frequency, therefore enhancing diffusion to the injured mitochondria.16 This is supported by the findings in this study that animals who received immediate fluid resuscitation had the greatest increase in CBF and overall cerebral perfusion; however, they were found to have brain lesion volumes and HIF-1α expressivity similar to DR animals (cerebral hypo-perfusion), likely due to the failure of fluid resuscitation alone to relieve the effects of the impaired micro-vasculature leading to inadequate microvascular perfusion and ultimately hypoxia after TBI + HS. In contrast, animals undergoing the low-frequency CBF oscillations delivered prior to fluid resuscitation, had significantly attenuated brain hypoxic injury, despite the lack of fluid resuscitation and delayed restoration of perfusion, suggesting an increase in the efficiency and efficacy of oxygen delivery as well as restoration of cerebral microvasculature perfusion.
Microvessel disruption plays a critical role in neuro-functional outcomes after TBI.39,40 The cerebrovascular endothelium, as a vascular barrier contacting blood directly, is dysfunctional following TBI.41,42 The endothelial nitric oxide (NO) generated in the cerebrovascular endothelium is one of the most important signaling molecules for CBF autoregulation and there are numerous studies that suggest it is neuroprotective after brain injury.43–45 Its production mainly depends on endothelial nitric oxide synthase (eNOS) activity, therefore, eNOS activation in the cerebrovascular endothelium plays a significant role in maintaining CBF and oxygenation after TBI, preserving brain microcirculation, inhibiting platelet aggregation, leukocyte adhesion and migration.46,47 Our results have shown that low-frequency CBF oscillation induced by TNS have upregulated mRNA expression of eNOS by 28% and 127% at 5 h and 24 h after TBI + HS respectively, while there was no significant change in both delayed and immediate resuscitation groups. This suggests that low-frequency CBF oscillations protected cerebral endothelial dysfunction in the peri-contusional areas from further injury that could be partially explained by oscillation-induced increase in vascular wall shear stress that plays a universal role in maintaining the integrity of the endothelium that lines the inner vascular wall.48–50
Regulation of CBF is closely related to neurological function.51–53 Recent studies from our lab26,54 and others have demonstrated that TNS can improve cerebral perfusion through the trigemino-cerebrovascular system. In the present study, we demonstrate that TNS-induced CBF oscillations at 0.1 Hz retains the improved CBF achieved after fluid resuscitation, leading to significantly improved neuronal function at 24 h after TBI + HS. The improved CBF that we observed in the animals that had received TNS-induced CBF oscillations is in agreement with previous reports of increased eNOS activity leading to improved CBF after TBI.55–57 In addition, many reports have shown that iNOS inhibition strategies are neuro-functionally protective possibly by stabilizing macro- as well as microcirculation.58–60 Our present results show that TNS-induced CBF oscillations also decreased iNOS expression. However, the exact mechanism of how CBF oscillations augment eNOS expression has not been elucidated. Further studies are necessary to explore this.
Besides brain ischemia and hypoxia, TBI + HS also induces inflammatory cascades which are one of the key drivers of worsening neurological outcomes.34 In this study, we show that brain tissue levels of TNF-α and IL-6 in peri-contusional areas were dramatically increased in both DR and IR animals after TBI + HS. However, the TNS-induced low-frequency CBF oscillation group showed significantly decreased levels of both cytokines, when compared with the animals under similar (DR) or shorter (IR) periods of cerebral ischemia. Generally, it is thought that ischemia triggers the expression of proinflammatory cytokines, subsequently attracting leukocytes into ischemic sites via induction of intercellular adhesion molecule (ICAM)-mediated leukocyte and the adhesion of leukocytes to the luminal wall of microvessels.61,62 Following adhesion, leukocytes migrate through the vessel wall into the brain parenchyma, triggering a major acute inflammatory response following brain injury.63–67 We clearly demonstrate that TNS-induced low-frequency CBF oscillations significantly decreased iNOS expression, TNF-α and IL-6 levels, and expression of ICAM1 in peri-contusional tissue (data not shown) despite ischemic conditions as compared to IR animals. These results indicate that the low-frequency CBF oscillations delivered after TBI + HS may not only protect the cerebrovascular endothelium by increasing eNOS expression, but also help to decrease capillary plugging and leukocyte adhesion in the peri-contusional brain tissue, where capillary stasis occurs and exaggerates these phenomena. Our results emphasize the importance of low frequency CBF oscillations beyond simply improving cerebral perfusion and oxygenation after TBI + HS.
Maneuvers such as body tilting and inspiratory resistance breathing have been used to elicit spontaneous CBF oscillations.17–20 These physiologic maneuvers take advantage of the body’s autonomic reactivity during periods of relative hypotension to enhance the body’s tolerance to hypotension; however, the physiologic response can be highly variable and inconsistent between individuals.68,69 In other words, not all subjects have the same degree of spontaneous CBF oscillations and therefore are not equally capable of tolerating hypotensive injury. Furthermore, these approaches would be difficult to apply to patients with TBI + HS. To overcome this, we demonstrate here that specific calibration of TNS parameters can consistently generate low-frequency oscillations in CBF, as seen in subjects that exhibit high tolerance to central hypovolemia. TNS is well-suited to induce CBF oscillations due to its rich network supplying a large portion of extrinsic neural supply to the blood vessels of the brain as well as its connections to various brainstem regions known to intrinsically modulate the cerebral microvasculature.22,25,26 Proximal to the Virchow-Robin space, the trigeminal nervous network, innervates the much of the cerebral vasculature.70–72 The results described herein show that TNS not only generated the controllable CBF oscillations in the targeted low frequency range, but also gradually improved the overall CBF amplitude. Given this evidence, it is reasonable to apply TNS as a tool to induce low-frequency CBF oscillations as a treatment strategy for TBI + HS.
This study has some limitations. First, animals were maintained under general anesthesia throughout the duration of the experiment. It is known that inhaled anesthetics, isoflurane in particular, exhibit neuroprotective properties that can confound our results.73 Additionally, there is evidence to suggest that isoflurane augments eNOS protein expression in animals.74 Furthermore, most clinical investigations of the physiology of spontaneous CBF oscillations have been performed in conscious subjects.17–20 While these limitations were minimized by sham, delayed and immediate resusciation experimental groups, the low frequency oscillation treatment of awake animals may represent another avenue of research to limit the effects of general anesthesia. Second, it should be noted that we proved our hypothesis that induced low-frequency CBF oscillations alleviate impaired cerebral microcirculation in peri-contusional brain tissue by using indirect measurements such as lesion volume, HIF-1α and eNOS expression, neuroinflammation, etc. It would have been ideal if the cerebral microcirculation were assessed directly using a capillary anemometer75 or in vivo two-photon laser scanning microscopy76 over the peri-contusional cortex. Third, most findings discussed herein represent outcomes at 5 h and 24 h after TBI + HS. However, long-term outcomes significantly influence behavior and higher cognitive function and have significant impact in the recovery from trauma.77,78 Future work should investigate whether the neuroprotective effects of induced CBF oscillations persist long-term as well. Finally, we have only studied outcomes with CBF oscillations at 0.1 Hz. In the future, we plan to study the effect of CBF oscillations at different frequencies.
In conclusion, our results demonstrate that TNS-induced low-frequency CBF oscillations at 0.1 Hz play a more significant role in preserving peri-contusional brain tissue than improving non-oscillatory cerebral perfusion or volume expansion in an animal model of severe TBI + HS. Our findings provide novel insights into the neuroprotective strategies employing low-frequency oscillations in CBF in injured brains to improve cerebral microcirculation and oxygenation. Although the experimental results shown in this study are promising, further studies are needed to understand the effects of CBF oscillations with different frequencies and to evaluate their effects on other organs.