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CHAPTER 3 proportion of total ED1+ cells contralateral (e) and ipsilateral (f) to the injury and example images from SCI (g) and SCI+670 (h) groups. (i-l): M2 (anti-inflammatory) microglia/macrophages (Arginase1+ED1+ double labelled) expressed as a proportion of total ED1+ cells contralateral (i) and ipsilateral (j) to the injury and example images from SCI (k) and SCI+670 (l) groups. All example images are taken from the injury zone of the dorsal horn at 7 days post-injury. Schematic cross section of spinal cord (bottom) indicates location of injury (dark grey penumbra) and region of quantification (light grey region). Scale bars: 50 μm. * p < 0.05 (linear mixed model); ** p < 0.01, *** p < 0.001 (Student’s t-test); † p < 0.05, †† p < 0.01, ††† p < 0.001 (Wilcoxon rank-sum). 3.5 Discussion Following spinal cord injury, 35 mW/cm2 of red (670 nm) light transcutaneously applied for 30 min/day for 7 days to the dorsal surface of rats is sufficient to reach the entire spinal cord and reduce the expression of pain behaviours. These reduced signs of allodynia are not due to sensorimotor deficits, as red light treatment improves both sensory and motor function. Alleviated hypersensitivity, improved tactile/proprioception (dorsal column) pathway functional integrity, and locomotor functional outcomes are preceded by reduced numbers of dying cells and reduced numbers of activated microglia/macrophages around the injury zone. Furthermore, the proportion of anti-inflammatory/wound healing (M2) microglia/macrophages is greatly enhanced by 24 hours following light treatment. The power output of the red light was sufficient to penetrate the entire rat spinal cord as red light could be seen with the naked eye illuminating through to the ventral surface of the cord in the sacrificed rat models. While penetration through to the rat spinal cord was achievable with an intensity of 35 mW/cm2, future studies would be required to determine the exposure parameters to achieve an equivalent level of irradiation in humans. The finding of 91% absorption (9% excess penetration) is a conservative measure for two main reasons: i) penetration measurements were obtained through the hair of unshaven sacrificed rats (the injury site of all injured animals was shaven), and ii) deoxygenated haemoglobin absorbs 670 nm significantly more than oxygenated haemoglobin (Meglinski and Matcher, 2002; Tin and Lal, 2015). Measurements from freshly scarified animals are therefore likely to have increased levels of deoxygenated blood, and thus reduced penetration, compared to live animals. Another factor to consider is the small attenuation of light as a function of distance from its source. The estimation indicates that over 93% of the light would have reached the spinal cord ventral surface if no intervening tissues were present to absorb the light, thus the effect of distance appears to be negligible. 9% excess penetration (i.e. 91% absorption) from the surface of skin (with hair intact) through all intervening tissue layers to the ventral surface of the spinal cord with a device delivering approximately 35 mW/cm2 is consistent with a recent study that demonstrated an excess penetration of 6.6% through the surface 72PDF Image | Effects of Red Light Treatment on Spinal Cord Injury
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