Introduction

Systemic complications associated with CHI are an important contributor to morbidity and mortality post injury. A bi-directional brain-systemic interaction plays a key role in maintaining physiological function through various mechanisms including HPA-axis. Trauma or injury can lead to primary endocrine failure with systemic alterations that can affect both neuronal as well as non-neuronal function¹. Additionally, activated immune system acts as an independent stimulus for endocrine and neuronal changes post TBI². Therefore, the study involved time-dependent assessment of serum metabolomics and inflammatory profile and how these changes influence serum corticosterone levels in the injured rats. Finally, to discern HPA axis function, changes in activation profile of hypothalamic microglial population was evaluated using morphological analysis.

Methods

Rats underwent mild and moderate weight-drop injury using modified Marmarou's weight drop model³. The trauma device consisted of a brass rod of 450g inside an acrylic tube (1 m) held in place by an iron rod. The brass rod was threaded to allow free fall due to gravity from desired height above the sagittal midway of the rat brain and were placed on an acrylic box filled with foam bed. The rats were divided into the following groups; Control group, Mild injury group that experienced free falling brass rod from a height of 25cm above the sagittal midway of the rat brain and Moderate injury group that experienced free falling brass rod from a height of 50cm. The serum and brain for immunohistochemistry were collected from rats, sacrificed post injury Day1 (Acute), Day5 (Sub-acute) and Day10 & Day30 (Chronic). Serum metabolite, inflammatory cytokine, corticosterone levels along with hypothalamic microglia were assessed at all timepoints. The NMR spectra of serum was acquired at 800.20 MHz, Bruker-AV spectrometer (Bruker, Germany) at 300K. Water suppressed Carr-Purcell- Meiboom-Gill (CPMG) spin echo pulse sequence (RD-90°-(τ-180°-τ) n acquire) with a total spin echo (2nτ) of 200ms was used. Spectral phase, baseline and chemical shift reference for each spectrum was corrected manually and the spectral region of 0.5–9.5ppm was integrated using TOPSPIN 3.7 (Bruker, Germany). Serum corticosterone and cytokine (IL-1β, TNF-α and IL-10) levels were measured using ELISA kits. Hypothalamic microglial morphology was assessed by fractal analysis using the FracLac plugin by the box counting method in FIJI.

Results

Metabolites including lactate, choline, leucine, isoleucine and valine were found as the classifiers that helped distinguish between mild and moderate groups during acute, sub-acute and chronic timepoints. The rats also showed distinct inflammatory profile where IL-1β and TNF-α levels were upregulated in moderate injured rats while IL-10 levels were downregulated in mild injured rats. Furthermore, injury specific alterations in serum metabolic and immunologic profile were found to be associated with hyperactive HPA axis, with consistent increase in serum corticosterone concentration post injury. Corticosterone concentration was increased at all timepoints in both injured groups except sub-acute timepoint of mild group. The hypothalamic microglia showed a characteristic activated de-ramified cellular morphology in both mild and moderate injured rats assessed using soma area, density, span ratio and fractal dimension of the microglial population.

Discussion

The present study has found that systemic alterations in the metabolite levels in the serum favored a subsequent decline in energy substrates and branched chain amino acids and this could be due to a concomitant increase in the levels of catabolic hormone CORT. Increased CORT production is a reliable mediator for HPA axis activity in the body and its persistent activation denotes a state of dysregulated HPA function. The impairment in the HPA activity is a consequence of increased microglial activity with de-ramified and amoeboid morphology in the hypothalamus of the injured rats. Along with these changes, serum inflammatory markers were found to be increased in moderate injured rats. Therefore, the study indicates a temporal state of systemic metabolic and immunological alteration post mild and moderate CHI. These systemic perturbations are associated with activation of hypothalamic microglial population and subsequent increased serum CORT levels.

Conclusion

The study suggests that HPA axis hyperactivity along with hypothalamic microglial activation led to temporal changes in the systemic metabolism and inflammation. These time dependent changes in the metabolite profile of rats can further strengthen the knowledge of diagnostic markers and help distinguish injury related outcomes after TBI.

Acknowledgements

This work was supported by Defence Research & Development Organisation (DRDO), Ministry of Defence, India (INM-324).