The Effects of Acute Lipopolysaccharide Induced Inflammation on Spinal Cord Excitability

Abstract

Peripheral inflammation alters the excitability of dorsal horn interneurons and increases flexor reflex strength (Dubner & Ruda, 1992); however, its effect on the spinal stretch reflex is not well understood. The stretch reflex is a muscle contraction in response to muscle stretch. We hypothesize that the acute inflammation caused by an injection of lipopolysaccharide (LPS) will cause an increase in spinal cord excitability. To test this hypothesis, we measured Hoffman’s (H) reflex, the electric analog of the stretch reflex in adult mice receiving an injection of LPS (.5mg/kg) or saline (200μl). Adult male and female mice (C57Bl/6) were anesthetized; then, the sciatic nerve was exposed and stimulated at current strengths from H-wave threshold (T) to 8T (20 x 0.1 ms pulses at 0.1 Hz). Recording electrodes were placed in the foot. We measured the maximum M wave amplitude (Mmax), maximum H wave amplitude (Hmax) and latencies of both waves. We compared the ratio of the maximal H wave over the maximal M wave (Hmax/Mmax), which reports the percentage of motor neurons activated by electrical stimulation of Group Ia muscle sensory neurons. Increased spinal cord excitability would be reflected in a larger Hmax/Mmax. We found that LPS-induced inflammation does not alter the Hmax/Mmax. While we found no evidence of changes in spinal cord excitability, inflammation could be altering Group Ia muscle spindle afferent responses to stretch. Future studies will test whether stretch reflex strength is altered by inflammation. Introduction Hoffman’s reflex (H-reflex) is an electrically induced reflex. The H reflex estimates of alpha motor excitability, which can be used to evaluate the response of the nervous system to different neurological conditions. The M wave is a contraction caused by direct stimulation of motor neuron axons and the H wave is derived from the reflex activation of the motor neurons by electrical stimulation of Group Ia afferents (Palmeri, Ingersoll, & Hoffman, 2004). 2 McNair Research Journal SJSU, Vol. 14 [2018], Art. 11 https://scholarworks.sjsu.edu/mcnair/vol14/iss1/11 DOI: 10.31979/mrj.2018.1411 142 Lipopolysaccharide (LPS) is a bacterial endotoxin found on the capsule of gram-negative bacteria (Gao et al., 2002). LPS is also known to cause an immune response in animals. Inflammation is synonymous with many neurodegenerative diseases (Qin et al,. 2004). When LPS is injected in an animal, cytokines, specifically TNFα, is released into the body (Qin et al., 2004). These cytokines cause a low level of chronic inflammation, much like a person gets when they catch the flu. Peripheral inflammation alters the excitability of dorsal horn interneurons and increases flexor reflex strength (Dubner & Ruda, 1992); however, its effect on the spinal stretch reflex is not well understood. My hypothesis is that LPS induced inflammation will increase spinal cord excitability; I expect to see an increase in HMax/MMax and earlier latencies in drug groups as compared to control groups. Additionally, I hypothesize that female mice will show an increase in spinal cord excitability as compared to males because females have a more robust immune system in response to bacterial infections (Klein, 2000). Methods C57/B16 adult (2-3 months) male mice were injected with lipopolysaccharide (LPS; 7.5 x 10 EU/kg in 200 μl saline) or control (200 μl saline) 18 hours before the experiment LPS. Mice were anesthetized with an intraperitoneal injection ketamine (100mg/kg) and xylazine (10mg/kg). The sciatic nerve was exposed and stimulating electrodes were placed around the sciatic nerve and recording electrodes are placed in the 4th dorsal interossei muscle of the foot (Figure 1). Electrical stimulations were induced to find threshold, the lowest voltage at which a stable H wave was elicited. Electrical stimulations were given at threshold and multiplied by 1.3, 1.5, 2, 3, 5, 6, 7 and 8T to find the maximum H-wave. Trains of 20 stimulations were given at 0.1 Hz. All data was recorded using LabChart. Hmax/Mmax is the ratio of peak amplitude of the H wave divided by the myotatic wave (M-wave). Amplitude of H and M waves were measured from peak to trough (Figure 2) (Turski, Bressler, Klockgether, & Stephens, 1990). Hmax/Mmax of LPS and controls were compared. Latency was measured from stimulation to start of each waveform (Figure 2) (Lee et al., 2009). The percentage of the motor neurons activated electrically was measured by normalizing the amplitude in millivolts of the H wave to the 3 Sanghera: The Effects of Acute Lipopolysaccharide Induced Inflammation on S Published by SJSU ScholarWorks, 2018 143 amplitude of the millivolts of the muscle contraction (M wave), with the amplitude of the M wave theoretically representing the maximum number of motor neurons that could be activated and the H wave the percentage of motor neurons that are actually activated. Figure 1: Description of the mouse anesthetized on a temperature controlled, heated surface with stimulating, recording and ground electrodes attached. Figure 2: Measurement of amplitudes and latencies in Hoffman’s wave. 4 McNair Research Journal SJSU, Vol. 14 [2018], Art. 11 https://scholarworks.sjsu.edu/mcnair/vol14/iss1/11 DOI: 10.31979/mrj.2018.1411 144 Results Hmax/Mmax ratio not significantly different between saline and lipopolysaccharide injected mice in either sex Using an in vivo method, the percent of motor neurons activated by electrical stimulation of the sciatic nerve is not significantly different between saline and lipopolysaccharide injected groups, p = .219 (Figure 3). There were also no differences between the sexes p = .905. M latency is unchanged by injection of LPS Figure 3: No changes in Hmax/Mmax ratios with LPS injection Hmax/Mmax ratio are not significantly different in SAL and LPS mice. Individual animal values are shown along with means and standard error of mean. M S A L