Chemo-neuropathy Evaluation Peripheral nerve amplitude and conduction velocity measurements <ul><li>Vincristine administered 2x / week (1.7 mg/kg sc) to mice for 10 weeks </li></ul><ul><li>Caudal (tail) nerve conduction velocity is increased by treatment </li></ul>Bieri et al, 1997, J. Neurosci. Res. 50:821-8 75 µV 2 ms
<ul><li>Conclusion : </li></ul><ul><li>Vehicle and veh/IGF treated animals showed a normal increase in caudal tail CV over 10 wks of treatment </li></ul><ul><li>Vincristine (Vin/Veh) treatment caused a reduction in CV over this time </li></ul><ul><li>The vincristine-induced decrease was ameliorated by IGF-I. </li></ul>IGF-I Protects Against Vincristine Reduction in Conduction Velocity (CV) Change in CV from pre-treatment baseline values
<ul><li>Gait measures (ipsi- and contralateral limb support) were reduced by vincristine treatment. IGF-I (1 mg/kg sc) reduced the effect of vincristine. </li></ul><ul><li>Hot plate latency was increased by vincristine treatment. The increase was prevented by IGF-I (1 mg/kg sc). </li></ul><ul><li>Axonal pathology (abnormal axons and myelin) produced by vincristine treatment was prevented by IGF-I (1 mg/kg sc). </li></ul><ul><li>Body weight was not affected by vincristine or IGF-I. </li></ul>Behavioral and Morphological Protection by IGF-I in Vincristine Chemoneuropathy
Conduction Velocity & Amplitude Changes Sural nerve .05 ms 10 mA Tibial (motor) distal proximal Sural (sensory) Caudal (mixed)
Nerve Conduction Velocities and Amplitudes at 5–7 Months of Age in SOD -/- Mice Wild type KO * * * Conclusion : SOD KO mice showed significant reductions in the conduction velocity of the caudal (tail) and tibial nerves, and in the latency of the plantar muscle response to tibial nerve stimulation.
Nerve Conduction in Adult SD Rats Sciatic notch Ankle 50 µ s 10 mA Tibial (motor) nerve recording x Tibial nerve Ave of 10 sweeps ISI: 2 sec Sciatic 100 0 -100 250 0 -250 Amplitude ( µV) 4.2 msec 6.8 msec Latency difference: (6.8 – 4.2) msec = 2.6 msec Distance: 40 mm Conduction Velocity: 40 mm / 2.6 msec = 15.4 m/sec Tibial 0 20 -10 10 Actual Data, Adult Rat
Nerve Conduction in Adult SD Rats 50 µ s 10 mA Sural (sensory) nerve recording Sural nerve x Amplitude ( µV) 0.75 msec Actual Data, Adult Rat Latency difference: 0.75 msec Distance: 23 mm Conduction Velocity: 23 mm / 0.75 msec = 31 m/sec 50 0 -100 50 0 2 6 -2 4 Ave of 10 sweeps ISI: 2 sec Stimulus artifact response
Nerve Conduction in Adult SD Rats Proximal Distal 200 0 -200 250 0 -250 Amplitude ( µV) 0 10 20 30 -10 3.0 msec 5.5 msec Actual Data, Adult Rat Latency difference: (5.5 – 3.0) msec = 2.5 msec Distance: 50 mm Conduction Velocity: 50 mm / 2.5 msec = 20 m/sec Ave of 10 sweeps ISI: 2 sec 50 µ s 10 mA 0 5 10 cm Proximal Distal Caudal (mixed) nerve recording
Spinal Reflex Excitability: C-fiber and Monosynaptic Reflexes
“ Early” response A /A β fibers “ Late” response C-fibers 100 200 300 0 400 Stimulus Time (msec) Plantar nerve Peroneus l. muscle Spinal cord Peroneal nerve Method for Recording Plantar A /A β and C-fiber Responses (CFR) C-fibers are small unmyelinated fibers transmitting diffuse pain signals A /A β fibers are larger myelinated fibers transmitting pain and touch information Hind foot 2 ms
“ early” 10 - 25 msec A /A β fiber response “ late” 150 - 400 msec C-fiber response Characterization of C-fiber Reflex (CFR) <ul><li>C-fiber response latency consistent with conduction in unyelinated C-fibers (0.5 - 1 m/sec) rather than myelinated A /A β fibers (12-20 m/sec) </li></ul><ul><li>Threshold of late response ~4x higher than early response </li></ul><ul><li>Capsaicin causes desensitization of late response consistent w/ C-fibers </li></ul>“ C-fibers ” are small unmyelinated axons mediating pain responses. They produce polysynaptic activation of spinal motoneurons and reflex muscle contractions – the “Late” response shown above.
Quantification of C-fiber reflex Average over 1 min Integrate over 225 msec Time from start (min) Amplitude (normalized %) 0 25 50 75 100 125 150 0 20 40 60 Integrated LHL Integrated RHL Integrated LHL Integrated RHL CFR Quantification Peroneal muscle EMG response Rectified Response 375 msec 150 375 t = 150 V i = V(t) dt 10 i = 1 V i CFR = / 10 6 sec 2 msec x 10 mA EMG Stimulus
Determination of afferent nerve pathway Determination of muscle of origin Verification of CFR Pathway The C-fiber response is produced by signals traveling in the plantar n. and activating motoneurons of the Peroneus L. muscle Peroneus l. muscle Tibialis anterior Biceps femoris (isolated) Soleus 100 msec Biceps (isolated) 100 msec Peroneus l. muscle response After transection of sural nerve 100 msec After transection of plantar nerve
Capsaicin 30 µl x 0.4 mg/ml at stimulation site Effect of Capsaicin on C-fiber Response Capsaicin initially enhances (6 & 12 sec) and then blocks the late response, consistent with desensitization of vanilloid receptors on C-fiber terminals. 18 s 24 s 30 s 36 s 42 s 48 s 54 s 6 s 12 s -5 s
Increased response at 3 mg/kg presumed to result from supra-spinal disinhibition relative to spinal inhibition Percent change in response Time relative to injection (min) -25 -15 -5 0 5 15 25 35 10 mg/kg 5.5 mg/kg 3 mg/kg PBS Morphine administered sc at time 0. N=3 rats per curve. Effect of Morphine on CFR Morphine produces a biphasic dose response effect on the C-fiber reflex, enhancing it at 3 mg/kg and suppressing it at higher doses. 0 20 40 60 80 100 120 140 160 180
Morphine-Induced Inhibition of CFR is Reversed by Naloxone Average CFR’s from R & L hind limbs in 1 rat 0 50 100 150 200 250 300 350 400 -20 -10 0 10 20 30 40 50 CFR amplitude, % baseline Morphine 10 mg/kg sc Naloxone 0.4 mg/kg sc Time relative to first injection (min) baseline * #
Determination of Site of Drug Action <ul><li>The earliest site of action would be at the P2X or related vanilloid receptors in the sensory nerve endings, which can be tested by binding or other in vitro studies. </li></ul><ul><li>Action of the drug on the unmyelinated axons in the sensory nerve can be identified by recording the amplitude of the compound (plantar) nerve action potential compared with the CFR amplitude. </li></ul><ul><li>The efferent pathway from the spinal cord to the plantar muscles can be tested by directly stimulating the peroneal nerve and recording the peroneus muscle (“M”) responses. </li></ul><ul><li>A spinal site of action can be evaluated by recording the dorsal horn field potential, which reflects the ability of the C-fiber afferents entering the cord to activate dorsal horn interneurons. </li></ul><ul><li>A supraspinal site of action can be evaluated by determining the effect of transection of the dorslal-lateral descending columns which modulate spinal excitability. </li></ul>Having established a model for evaluating analgesic effects of drugs via changes in the C-fiber reflex, the site of drug action needs to be identified.
Peroneal muscle EMG Plantar nerve Peroneus l. muscle Hind foot stimulation 2 ms 14 0 mA Spinal cord Plantar nerve afferent volley Conduction velocity = 0.5 - 1.0 m/s Integration window Plantar n. Afferent Volley versus CFR 50 msec 0 20 40 60 80 100 0 3 6 9 12 15 Stimulus current (mA) Integrated EMG / CAP (% max.) Peroneal m. EMG Plantar n. APV Stimulus-Response Recruitment
Test Agent Does Not Inhibit Plantar Nerve C-fiber Afferent Volley Mean effect of test agent Effect of test agent vs. time Percent change in response CFR Plantar n. APV 0 1000 2000 3000 -20 -10 0 10 20 Integrated activity Peroneus l. muscle EMG Plantar n. volley 4000 Time relative to injection (min.) Test agent 3 mg/kg i.v. The C-fiber response but not the amplitude of the plantar n. volley is reduced by the test drug => the drug is not acting on the efferent pathway. 0 20 40 60 80 100 120 Veh. Test agent Veh. Test agent p=0.013 p>0.05 N=4 N=4
Effect of Test Agent on the Efferent Peroneal Neuromuscular Pathway 0 100 200 300 400 500 600 -40 -20 0 20 40 Peroneal muscle amplitude Hind foot stimulated C-fiber response (mv*msec) Peroneal n. direct M-response (mV, 25x) Time (min) post injection -42 4 -26 24 2 msec Time (min) relative to test agent injection (3 mg/kg iv) 100 msec M-response C-Fiber Response The direct M response is not effected by the test drug => drug is not acting on the efferent path. Plantar nerve 2 ms 10 mA Spinal cord Peroneus l. muscle EMG Peroneal nerve .05 ms 10 mA
Chronic Dorsal-lateral Funiculus (DLF) Lesion and CFR The test agent blocked the CFR in normal animals (not shown), and also blocked it in animals with chronic DLF lesions. Chronic DLF lesions were made in rats ~4 weeks prior to evaluation of a test agent on the CFR. Spinal lesions did not block the response to morphine or naloxone (not shown). Lesion of the DLF pathway does not block CFR inhibition produced by test agent => drug does not act at supraspinal level. T9 cord DLF lesion Test Agent 3 mg/kg iv. 0 2000 4000 6000 8000 -20 -10 0 10 20 30 40 Time post injection (min) Integrated EMG activity CFR Amplitude (mv*ms/100) 0 40 80 120 160 Pre injection 15’ Post injection 62.5% p<0.0001 N= 10
Peroneus l. muscle 100 msec Plantar nerve 2 ms 14 mA L4 Spinal cord Peroneus l. muscle EMG myelinated afferent response C-fiber DHEP: DHEP amplitude: Hind foot stimulation 10x gain Peroneal nerve Spinal Cord Dorsal Horn Field Potentials Plus CFR Recording
Test Agent Does Not Inhibit Dorsal Horn Evoked Potential Effect of test agent vs. time Mean response inhibition by test agent 0 20 40 60 80 100 120 140 -10 0 10 20 30 40 50 % change in amplitude CFR vs. DHEP C-fiber refles Dorsal horn evoked potential Time from injection (min) Test agent 3 mg/kg iv. Percent inhibition 80 60 40 20 0 CFR amplitude DHEP amplitude N=3 p< 0.05 N.S. The test drug does not reduce the amplitude of the dorsal horn evoked potential => the drug does not impair transmission between primary efferent terminals and the first-order spinal interneurons in the dorsal horn.
Spinal Reflex Excitability: Spinal Monosynaptic (H-) Reflex <ul><li>The Hoffman or “H” reflex is the monosynaptic muscle reflex produced by activating proprioceptive muscle afferents; aka the common achilles tendon-tap reflex </li></ul><ul><li>Stimulation of the tibial nerve activates axons innervating the plantar muscle, producing a direct “M” or muscle response, and also proprioceptive afferents traveling to the spinal cord, which then activate spinal motoneurons producing a second muscle response. </li></ul><ul><li>Unlike the CFR, the H-reflex does not directly involve any excitatory or inhibitory interneurons. Thus drugs that affect e.g. GABA receptors or release should not affect this reflex unless (like GABA-A agonists) they tonically increase GABAergic tone, whereas they do impair the C-fiber reflex. </li></ul>Proprioceptive afferents 0.5 ms 1-10 mA Spinal cord Hind foot Tibial nerve Spinal interneurons Motor neurons Plantar muscle Muscle (“M”) response Monosynaptic (“H”) response DRG
Characterization of the Plantar H- (Monosynaptic) Reflex <ul><li>Stimulation of the tibial nerve produces a direct muscle (M) response in the plantar muscle starting about 3 msec after the stimulation, followed by an H (monosynaptic) reflex response at about 10 msec. </li></ul><ul><li>GABA-A receptor agonist drugs typically reduce this response, while antagonists facilitate it, assuming the drugs penetrate the blood-brain barrier. Benzodiazepines typically have no effect. </li></ul><ul><li>A drug that directly affects peripheral axons or neuromuscular junctions (e.g. ssuccinylcholine) should inhibit this reflex. </li></ul>-10 -5 0 5 10 15 Time (ms) EMG (mV) M-response H-reflex stimulus
H- or monosynaptic reflex (MSR) responses from rat at various times before and after injection of either vehicle or 0.5 mg/kg IV diazepam. Each waveform is the average of 10 successive responses obtained at 6 sec intervals. Red biphasic square wave at time 0 represents stimulus pulse. Scale at bottom right in mV applies to all recordings. Diazepam, a benzodiazepine, has no effect on monosynaptic reflexes. (Lack of) Effect of Diazepam on H-Response 0 200 400 600 800 1000 1200 1400 -20 0 20 40 60 80 100 Time (min) Peak-Peak Amplitude ( µV) M response Vehicle H response Diazepam 0.5 mg/kg IV 10 min before Vehicle inject. Time of Vehicle inject. 10 min before Drug inject. Time of Drug inject. 10 min after Drug inject. 20 min after Drug inject. 30 min after Drug inject. Time (msec) -5 0 5 10 15 0 2.0 4.0 -4.0 -2.0 -6.0 MSR Amplitude M response H response
Assessment of Spinal Cord Function Magnetic Motor Stimulation: Basic Principles and Clinical Experience (EEG Suppl. 43; chapter 25, pps. 293-307
SEP ASR Motor function Somatosensory Evoked Potentials Auditory Stimulated Responses Cerebellar Myoelectric Evoked Responses Conclusion : Sensory and motor evoked potentials provide a reliable means of monitoring recovery after spinal injury.
Auditory Sensory Gating Responses Effect of Amphetamine
<ul><li>Paradigm : </li></ul><ul><li>Electrodes implanted in rats under sodium pentobarbital anesthesia: </li></ul><ul><ul><li>Left frontal cortex - left sensory-motor cortex (above hippocampus) </li></ul></ul><ul><ul><li>Depth electrode, right CA3 region of the HC, referenced to a skull screw </li></ul></ul><ul><ul><li>Neck EMG </li></ul></ul><ul><li>One week after recovery, animal exposed to auditory tones as follows </li></ul><ul><ul><li>Pairs of 5 k Hz tones, 10 ms duration, 0.5 s apart </li></ul></ul><ul><ul><li>10 s interval between pairs of tones </li></ul></ul><ul><li>Outcome: </li></ul><ul><ul><li>Amplitude = P1 - N1, mV (most robust effect) </li></ul></ul><ul><ul><li>Outcome = ratio of amplitude of second (test) to first (conditioning) response. </li></ul></ul>Evaluation of Attention by Auditory Sensory Gating Response Stereotaxically placed electrodes 4.0 mm below dura in the hippocampal CA-3 region skull
Effect of Amphetamine on Auditory Gating Responses <ul><li>Rats were chronically implanted with screw electrodes over frontal and sensory-motor cortices, and with a bipolar metal electrode into the CA3 region of the hippocampus (electrode tip separation ~ 1 mm). </li></ul><ul><ul><li>- Test tones were applied during surgery to optimize electrode placemnt </li></ul></ul><ul><li>Post surgical recovery, animals were placed into recording chamber and exposed to paired tones: </li></ul><ul><ul><li>- 3 k Hz, 10 ms duration </li></ul></ul><ul><ul><li>- 0.5 s interval between test tones </li></ul></ul><ul><ul><li>- 10 s between pairs of test tones </li></ul></ul><ul><li>Three sets of 30 stimulus tone pairs were delivered at ~ 6 min intervals while the rat was awake and resting </li></ul><ul><li>Amphetamine (1 or 3 mg/kg ip) was then administered </li></ul><ul><li>10’, 20’, and 30’ post drug administration, additional sets were recorded. </li></ul><ul><li>Individual peak amplitudes were analyzed and compared as a function of “Conditioning” vs “Test” tone pulses, and drug: “Pre” vs “Amphetamine”. </li></ul>
Effect of Amphetamine on Auditory Gating Responses <ul><li>P0 not well defined in EEG records, but N1 and P1 were </li></ul><ul><li>P0, N1, and P1 were well defined in CA-3 records </li></ul><ul><li>In both surface and CA3 recordings, identified potentials occurred at similar latencies in both Conditioning and Test responses </li></ul><ul><li>P0 and N1 were slightly delayed from EEG to CA3 (~ xx ms), while P1 and N1 showed similar latencies: Are the potentials analagous?? </li></ul>Analysis of Peak-Peak Amplitudes of Auditory Evoked Potentials Mean latencies (N= 3 responses) for the (P1 - N1) amplitude difference as a function of (conditioning vs test) and (Pre drug vs Amphetamine). P1-N1 0.0 0.5 1.0 1.5 2.0 2.5 Pre Drug Amphetamine 1 mg/kg IP Amplitide (mV) Cond. Test (Cond vs Test): ANOVA, p= 0.02 V C V T
Analysis of percent inhibition of the test tone for various amplitude measures 0 20 40 60 80 100 P0-N1 P1-N1 P0-N1+P1 % Inhibition Pre drug Amphet. * * * p= 0.023 p= 0.009 p= 0.008 unpaired t-test, N= 3 100% = complete inhibition; 0% = no effect Amphetamine reduced inhibition of the Test evoked potential by all measures, with P1-N1 and P0-N1+P1 showing the most robust effect. Effect of Amphetamine on Auditory Gating Responses Evoked Potential Peak-Peak Measure % Inhibition = (V C – V T ) * 100 V C
Effect of Amphetamine on Gating Responses Post Amphetamine 1 mg/kg IP -0.8 -0.4 0.0 0.4 0.8 20 40 60 80 100 120 140 Time (ms) N1 P1 Amplitude (mV) Amphetamine at both 1 and 3 mg/kg IP reduced inhibition of the auditory evoked gating responses. Pre Drug -0.8 -0.4 0.0 0.4 0.8 20 40 60 80 100 120 140 Time (ms) Amplitude (mV) N1 P1 Conditioning Test Tone 0 20 40 60 80 100 1.0 3.0 Amphetamine (mg/kg ip) Percent inhibition of Test Response Pre dosing Post dosing p< 0.001 p= 0.001 N=9 N=5 N = # of rats; P1-N1 amplitudes Conditioning Test