Miniature Broadband-NIRS System to Measure CNS Tissue Oxygenation and Metabolism in Preclinical Research

In-vivo measurement of CNS tissue oxygenation and metabolism is critical in health and disease. Broadband-near infrared spectroscopy is a non-invasive optical technique which measures tissue oxygenation, haemodynamics and metabolism through in-vivo quantification of concentration changes of oxy- and deoxy-haemoglobin (Δ[HbO2] and Δ[HHb]) and oxidised cytochrome-c-oxidase (Δ[oxCCO]). Current commercially available NIRS systems only use a few wavelengths to measure concentration change that fails to provide accurate Δ[oxCCO] measurement. Broadband-NIRS instruments however, use more than 100 wavelengths which enables quantification of change in [oxCCO], an important marker of cellular oxidative metabolism. These systems tend to be bulky, requiring extensive calibrations and trained staff to operate them; making them less versatile and difficult to be adapted in the clinical environment. Furthermore, existing broadband-NIRS systems quantify chromophore concentration changes assuming a fixed optical pathlength across all the subjects using a previously measured DPF (differential pathlength factor) with time or frequency domain systems. This thesis describes the development of a portable broadband-NIRS system called mini-CYRIL “CYtochrome Research Instrument and appLication”, based on easily sourced components. A miniature white light source (HL-2000-HP) and miniature spectrometers (QE65pro and Ventana VIS-NIR) by Ocean Optics were customised for measuring CNS tissue oxygenation and metabolism. While having the features of commercially available NIRS systems in terms of portability, ease of use and no need for wavelength calibration, in terms of performance mini-CYRIL is comparable to broadband-NIRS instruments providing reliable Δ[oxCCO] measurements that have been validated and assessed through in-vivo tissue studies in (a) preclinical model of: (i) neonatal hypoxic-ischaemic (HI) encephalopathy, (ii) multiple sclerosis (MS) and (iii) low-light level therapy in the aged retina; (b) infants during brain functional activation. Mini-CYRIL is furthermore novel in offering calculation of absolute change in the concentration of chromophores based on real-time measurement of the optical path of light traversing the tissue. None of the current NIRS systems offer this feature which is crucial in case of changing pathology following an injury

Watching the human retina breath in real time and the slowing of mitochondrial respiration with age

The retina has the greatest metabolic demand in the body particularly in dark adaptation when its sensitivity is enhanced. This requires elevated level of perfusion to sustain mitochondrial activity. However, mitochondrial performance declines with age leading to reduced adaptive ability. We assessed human retina metabolism in vivo using broad band near-infrared spectroscopy (bNIRS), which records colour changes in mitochondria and blood as retinal metabolism shifts in response to changes in environmental luminance. We demonstrate a significant sustained rise in mitochondrial oxidative metabolism in the first 3 min of darkness in subjects under 50 years old. This was not seen in those over 50 years. Choroidal oxygenation declines in  50 s. Significant group differences in blood oxygenation are apparent in the first 6 min, consistent with mitochondrial demand leading hemodynamic changes. A greater coupling between mitochondrial oxidative metabolism with hemodynamics is revealed in subjects older than 50, possibly due to reduced capacity in the older retina. Rapid in vivo assessment of retinal metabolism with bNIRS provides a route to understanding fundamental physiology and early identification of retinal disease before pathology is established

The Role of Neuroglobin in Retinal Hemodynamics and Metabolism: A Real-Time Study

Purpose: In this study, we used broadband near-infrared spectroscopy, a non-invasive optical technique, to investigate in real time the possible role of neuroglobin in retinal hemodynamics and metabolism. / Methods: Retinae of 12 C57 mice (seven young and five old) and seven young neuroglobin knockouts (Ngb-KOs) were exposed to light from a low-power halogen source, and the back-reflected light was used to calculate changes in the concentration of oxygenated hemoglobin (HbO2), deoxygenated hemoglobin (HHb), and oxidized cytochrome c oxidase (oxCCO). / Results: The degree of change in the near-infrared spectroscopy signals associated with HHb, HbO2, and oxCCO was significantly greater in young C57 mice compared to the old C57 mice (P < 0.05) and the Ngb-KO model (P < 0.005). / Conclusions: Our results reveal a possible role of Ngb in regulating retinal function, as its absence in the retinae of a knockout mouse model led to suppressed signals that are associated with hemodynamics and oxidative metabolism. / Translational Relevance: Near-infrared spectroscopy enabled the non-invasive detection of characteristic signals that differentiate between the retina of a neuroglobin knockout mouse model and that of a wild-type model. Further work is needed to evaluate the source of the signal differences and how these differences relate to the presence or absence of neuroglobin in the ganglion, bipolar, or amacrine cells of the retina

Dexmedetomidine Combined with Therapeutic Hypothermia Is Associated with Cardiovascular Instability and Neurotoxicity in a Piglet Model of Perinatal Asphyxia

The selective α2-adrenoreceptor agonist dexmedetomidine has shown neuroprotective, analgesic, anti-inflammatory, and sympatholytic properties that may be beneficial in neonatal encephalopathy (NE). As therapeutic hypothermia is only partially effective, adjunct therapies are needed to optimize outcomes. The aim was to assess whether hypothermia + dexmedetomidine treatment augments neuroprotection compared to routine treatment (hypothermia + fentanyl sedation) in a piglet model of NE using magnetic resonance spectroscopy (MRS) biomarkers, which predict outcomes in babies with NE, and immunohistochemistry. After hypoxia-ischaemia (HI), 20 large White male piglets were randomized to: (i) hypothermia + fentanyl with cooling to 33.5°C from 2 to 26 h, or (ii) hypothermia + dexmedetomidine (a loading dose of 2 μg/kg at 10 min followed by 0.028 μg/kg/h for 48 h). Whole-brain phosphorus-31 and regional proton MRS biomarkers were assessed at baseline, 24, and 48 h after HI. At 48 h, cell death was evaluated over 7 brain regions by means of transferase-mediated d-UTP nick end labeling (TUNEL). Dexmedetomidine plasma levels were mainly within the target sedative range of 1 μg/L. In the hypothermia + dexmedetomidine group, there were 6 cardiac arrests (3 fatal) versus 2 (non-fatal) in the hypothermia + fentanyl group. The hypothermia + dexmedetomidine group required more saline (p = 0.005) to maintain blood pressure. Thalamic and white-matter lactate/N-acetylaspartate did not differ between groups (p = 0.66 and p = 0.21, respectively); the whole-brain nucleotide triphosphate/exchangeable phosphate pool was similar (p = 0.73) over 48 h. Cell death (TUNEL-positive cells/mm2) was higher in the hypothermia + dexmedetomidine group than in the hypothermia + fentanyl group (mean 5.1 vs. 2.3, difference 2.8 [95% CI 0.6-4.9], p = 0.036). Hypothermia + dexmedetomidine treatment was associated with adverse cardiovascular events, even within the recommended clinical sedative plasma level; these may have been exacerbated by an interaction with either isoflurane or low body temperature. Hypothermia + dexmedetomidine treatment was neurotoxic following HI in our piglet NE model, suggesting that caution is vital if dexmedetomidine is combined with cooling following NE

Nimodipine reduces dysfunction and demyelination in models of multiple sclerosis

Objective: Treatment of relapses in multiple sclerosis (MS) has not advanced beyond steroid use, which reduces acute loss of function, but has little effect on residual disability. Acute loss of function in an MS model (experimental autoimmune encephalomyelitis; EAE) is partly due to CNS hypoxia, and function can promptly improve upon breathing oxygen. Here we investigate the cause of the hypoxia and whether it is due to a deficit in oxygen supply arising from impaired vascular perfusion. We also explore whether the CNS‐selective vasodilating agent, nimodipine, may provide a therapy to restore function, and protect from demyelination in two MS models. Methods: A variety of methods have been employed to measure basic cardiovascular physiology, spinal oxygenation, mitochondrial function and tissue perfusion in EAE. Results: We report that the tissue hypoxia in EAE is associated with a profound hypoperfusion of the inflamed spinal cord. Treatment with nimodipine restores spinal oxygenation and can rapidly improve function. Nimodipine therapy also reduces demyelination in both EAE, and a model of the early MS lesion. Interpretation: Loss of function in EAE, and demyelination in EAE and the model early MS lesion, appear to be due, at least in part, to tissue hypoxia due to local spinal hypoperfusion. Therapy to improve blood flow not only protects neurological function, but also reduces demyelination. We conclude that nimodipine could be repurposed to offer substantial clinical benefit in MS