NIH NS43798 (DMB); NIH F31 NS058138 (MRD); P30-NS045758 (CBSCR); and the Paralyzed Veterans of America #2451 (DMB) and #2707 (MRD).

1. Up/Down Plantar Von Frey Testing
The purpose of the test is to determine tactile sensitivity by measuring how much force is required to elicit paw withdrawal from the stimulus. Commercially available calibrated Von Frey Hair monofilaments between the ranges of 4.56 and 6.10 are used to stimulate the paw. Other sensory input like visualization of the stimulus or proprioception can confound tactile threshold measures so responses based on visual or proprioceptive input are invalid. To prevent visualization of the monofilament touching the skin, we test only while the rat is eating cereal. This cognitive distracter, also ensures that all rats are tested at similar baseline levels of comfort. To avoid responses due to proprioceptive rather than tactile input, we do not include trials where the monofilament lifts or moves the paw.
A. ACCLIMATION PROCEDURES FOR THE UP-DOWN APPROACH
<ol>
	<li>Present fruit cereal in the home cage so rats recognize and seek the food reward. Rats do not need to be food deprived at any point in the gentling or testing process.</li>
	<li>Rats are ready to be acclimated to the testing environment and procedures once they eat fruit cereal in the home cage. Acclimation occurs over at least 4 sessions of 10 minutes each but more may be needed. Ten minute sessions are used so that the rats do not become satiated before the end of the session.</li>
	<li>Place the rat in the testing chamber with three pieces of fruit cereal. Provide additional fruit cereal one to two at a time as they are eaten. On the third session while the rat is eating, produce hand movements under the wire floor so that the rat becomes accustomed to movement below its paw. On the fourth session, apply the 5.18 monofilament to the plantar surface of the paw approximately once a minute for a total of ten presentations. A na&iuml;ve rat should not respond to this sub threshold stimulus.</li>
	<li>Tally the pieces of cereal consumed, frequency of urination and defecation, freezing, and grooming behaviors. Freezing behavior occurs in two ways: a) the rat is crouched low to the ground usually near the back wall or facing away from the examiner and remains motionless; b) the rat is hunched over in the middle of the cage and usually chatters its teeth. Freezing behaviors may continue for long periods of time.</li>
	<li>A gentled rat that is ready for testing will eat nearly continuously for 10 minutes. It will move easily in the cage and seek fruit cereal very quickly after consuming the last. Importantly, the rat will not stop eating or be distracted by hand movements under the wire floor or monofilament application to the paw.</li>
</ol>
B. TESTING PROCEDURES FOR THE UP-DOWN APPROACH
<ol>
	<li>Rats produce distress vocalizations that are above our human aural range. Rats waiting to be tested should be kept in a separate room so that they can not hear these vocalizations.</li>
	<li>Rats with spinal cord injury that are appropriate for up-down von Frey Hair testing must demonstrate weight bearing with the limbs. Limbs that are unable to support the rat cannot be reliably tested because the application of monofilaments moves the paw before the hair bends.</li>
	<li>Place a rat in the testing chamber with 2 or 3 pieces of fruit cereal. Rats must be eating during each VFH application.</li>
	<li>Roll a die to determine which HL to start testing first. Even numbers represent the right paw and odd numbers represent the left.</li>
	<li>Begin the testing session with the 5.18 monofilament. Withdrawal responses can occur from 4.56 to 6.10 in normal and SCI rats and the 5.18 monofilament falls in the middle of the range.</li>
	<li>Do not apply a monofilament while a rat freezes, crouches or does not eat. If the tester waits, sometimes these conditions will resolve and testing can resume. If the rat displays these behaviors and the total time in the apparatus is 20 minutes, stop the testing and retest from the beginning the next day. Do not remove the rat while it is displaying these behaviors as this may increase their occurrence. Wait until the rat breaks out of these patterns before handling it to avoid reinforcing these behaviors.</li>
	<li>Touch the monofilament to the L5 plantar dermatome of the hind paw between the foot pads until it bends (see Figure 1A). This smooth steady application should take about one second then remove it from the skin. Avoid touching any part of the foot pads as the sensitivity of the skin is much lower in these areas. This single touch constitutes one trial.</li>
	<li>A positive response occurs when the rat quickly lifts the paw up away from the monofilament. The withdrawal must be initiated by the time the monofilament is removed in order to confirm that the response is associated with the monofilament application.</li>
	<li>Watch the animal for other evidence of awareness of the monofilament touch such as vocalization, looking downward, backward or at the tested paw, licking the paw, sustained paw withdrawal or twitching the ears. Note them on the data sheet. When these behaviors occur with paw withdrawal it serves as evidence of supraspinal perception of the stimulus.</li>
	<li>If there is a positive response, apply the next lowest monofilament. If there is no response (negative), move up to the next higher monofilament. Allow 30 seconds between each monofilament application for the sensory receptors to come back to baseline firing before retesting (to prevent adaptation).</li>
	<li>Continue applying higher or lower monofilaments to the hind paw based on positive or negative responses until you have 10 trials. Then repeat on the other hind paw. (see example datasheet).</li>
	<li>The highest VFH that can be tested without introducing proprioceptive input and lifting the paw is 6.10. The 6.45VFH and larger should not be used because the paw lifts before the monofilament bends. If there is no response at 6.10 then the next trial will apply the 6.10 again.</li>
	<li>A response should be voided and the trial must be re-tested if one of the following occurs during the stimulus application or paw withdrawal:
	<ol class="lalpha">
		<li>The rat is not continuously eating.</li>
		<li>The rat moves away from the VFH stimulus prior to the bending of the monofilament, making it impossible to know the force applied that elicits the withdrawal response.</li>
		<li>The paw is lifted by the VFH (either before or after the VFH is bent) providing proprioceptive in addition to tactile stimulation.</li>
	</ol>
	</li>
	<li>A testing session usually can be completed in less than 15 minutes for both hindlimbs.</li>
</ol>
C. DATA PROCESSING FOR THE UP-DOWN APPROACH
<ol>
	<li>A successful test will have 10 sequential trials for each paw.</li>
	<li>The value of the tactile threshold is the lowest monofilament that produces paw withdrawal on &ge;50% of the presentations and 3 or more presentations of the monofilament have be given (see example datasheet, thresholds are circled).</li>
	<li>A monofilament with only 2 total trials is not selected as the threshold even when one trial is positive and one is negative. Multiple presentations of the stimulus are better indicators of the pattern of responses to that force.</li>
	<li>If there is no monofilament that produces positive responses on at least 50% of the tests, the rat is given a score of 6.45. This means that the rat did not detect the highest testable monofilament of 6.10 beyond random chance. A value of 6.45 is given because it represents the next value higher than the testable range of monofilaments.</li>
	<li>Convert the threshold value for the right and left paws into gram force. The gram force values will be provided by the supplier of the von Frey hairs. . For data from bilateral injuries, the threshold gram force for the two paws can be averaged together to yield a single score.</li>
</ol>
2. Dorsal Von Frey Testing
The purpose of this test is to determine tactile sensitivity in rats with such poor motor control that they cannot be tested using the typical plantar VFH methods. It quantifies how much touch force is needed to produce an aversive response. To avoid responses to proprioceptive stimuli, we do not include trials in which the paw is moved by the VFH during application. Additionally, we do not include trials in which the VFH slips or is brushed across the dorsal surface of the foot.
A. ACCLIMATION PROCEDURES FOR DORSAL VON FREY TESTING
<ol>
	<li>Gentle rats for four sessions of 10 minutes each prior to baseline testing and spinal cord injury.</li>
	<li>Loosely wrap the rat in a small towel so the hindquarters are exposed as shown below (Figure 1B). Position the rat over a table with the hind paws lightly resting on the surface. Use the same snug but not tight wrap and hold for each rat. Bracing your forearm on the table edge may reduce variability between rats.</li>
	<li>A gentled rat that is ready for testing will not resist being held in the towel for several consecutive minutes; however you may have to re-wrap the rat 1 or 2 times during the testing of both hind paws.</li>
</ol>
B. TESTING PROCEDURES FOR DORSAL VON FREY TESTING
<ol>
	<li>Roll a die to determine which hind limb to start testing first. Even numbers represent the right paw and odd numbers represent the left.</li>
	<li>Loosely wrap the rat in the towel leaving the hindquarters exposed.</li>
	<li>Tactile stimulus is applied only after the rat is calm and quiet for at least 10 seconds.</li>
	<li>Starting with the 4.56 VFH, apply the monofilament perpendicularly to the dorsal surface in between the first and second metatarsal approximately 1 cm proximal to the joint (Figure 1C). Apply the monofilament in a smooth, steady manner over a one second interval until it bends then remove it. This single touch constitutes one trial.</li>
	<li>A positive response occurs when the rat quickly moves the paw away from the stimulus. The direction of motion is often posterior or external before the hind limb is flexed. The withdrawal must be initiated by the time the monofilament is removed in order to confirm that the response is associated with the monofilament application.</li>
	<li>Apply the 4.56 monofilament twice more with at least 30 seconds between applications in order to prevent sensory "windup" and to allow the sensory receptors to come back to baseline firing before retesting (to prevent adaptation).</li>
	<li>A stimulus test should be reapplied if the VFH slips and gently grazes the skin, making the force delivery more of a brushing or a sweep rather than a pointed contact.</li>
	<li>If there are fewer than 2 (i.e. 1 or 0) positive responses to the 4.56 monofilament, the next larger VFH is applied 3 times in the same manner.</li>
	<li>In this way, progressively larger VFHs from 4.56 through 5.88 are applied until two or more positive responses occur or until the largest VFH is reached.</li>
	<li>The paw withdrawal threshold is the lowest VFH which elicits at least 2 positive responses out of 3 trials.</li>
	<li>Repeat on the other paw.</li>
	<li>A testing session usually can be completed in 15 minutes for both hind paws.</li>
</ol>
3. Representative Results
A successful test will result in immediate, brisk withdrawal of the hind paw to a perpendicularly applied VFH to the mid-plantar region (Figure 1A) or the dorsal surface between the 1st and 2nd metatarsal 1 cm from the joint (Figure 1C). These responses are tallied to derive tactile threshold for each hind paw. Tactile thresholds from dorsal VFH tests in acute SCI showed excellent fidelity with plantar VFH thresholds derived at chronic times (Figure 2). Declines in tactile thresholds derived from plantar or dorsal tests occur for graded SCI (Figure 33,5). This hypersensitivity manifests as early as 7 days after SCI and is maintained for the duration of the experiment3-4,6. While a range of SCI severities do not affect tactile sensory thresholds of the paw, declines in thresholds occur for more severe SCI (Figure 3). Importantly, the development of below-level allodynia is an all-or none response that occurs when there is a loss of more than 60% of the white matter at the lesion epicenter. (Figure 43).
Tactile thresholds can decay and produce artificial responses to low VFH forces. At least two conditions can produce threshold decay - rapidly applying the VFH to the skin in a poking or jabbing manner or not waiting long enough between stimulus applications. To be confident that the tactile thresholds are genuine, care must be taken to apply each VFH using a similar low velocity approach and the interstimulus interval must be at least 30 sec. We have also shown that eating behavior reduces threshold decay5.
<img alt="Figure 1" src="/files/ftp_upload/3247/3247fig1.jpg" /> 
Figure 1. Schematic and photograph depicting the location of VFH stimulation for the plantar and dorsal von Frey hair (VFH) testing. A) For plantar VFH testing, the von Frey hair should be applied perpendicularly to the mid-plantar hindpaw approximately 1 cm posterior to the middle phalange (as indicated by the red box in the diagram of the plantar surface of the hindpaw) at a slow consistent speed until it bends. B and C) For the dorsal VFH technique, the rat is loosely wrapped in a surgical towel as shown in the photograph. The VFH is applied to the dorsal surface of the paw in between the first and second metatarsal approximately 1 cm proximal to the joint.
<img alt="Figure 2" src="/files/ftp_upload/3247/3247fig2.jpg" /> 
Figure 2. Dorsal von Frey Hair sensory thresholds correlate to standard plantar VFH. (n-44; Pearson Coefficient = 0.503, p &lt; 0.01, r2 = 0.67). Dashed lines show the cutoff threshold for allodynic sensation using either the Dorsal VFH or the plantar VFH techniques and indicate that allodynic thresholds at 7 days after SCI using the Dorsal VFH test predict chronic below-level allodynia determined by plantar VFH methods.
<img alt="Figure 3" src="/files/ftp_upload/3247/3247fig3.jpg" /> 
Figure 3. Early dorsal von Frey Hair sensory thresholds predict allodynic and non-allodynic tactile sensitivity across injury severities at later timepoints. Comparable sensory thresholds occurred between 7 days post SCI Dorsal VFH and 28 days post SCI plantar VFH measures. Remarkable similarity occurred for a wide range of SCI, with significant allodynia noted in moderate to severe SCI (p&lt;.05 1.1 mm displacement vs. all other groups).
<img alt="Figure 4" src="/files/ftp_upload/3247/3247fig4.jpg" /> 
Figure 4. Percentage of rats receiving laminectomy (no SCI) and SCI groups showing hindpaw allodynia at 6 weeks after SCI. Allodynia was only seen in rats with moderate or severe SCI (OSU ESCID device 0.9, 1.1, 1.25, 1.3 mm cord displacements) while rats with mild SCI (0.3, 0.5, 0.7 mm spinal cord displacements) exhibited normal responses to tactile stimulation. Representative coronal spinal cord sections stained for myelin at the lesion epicenter for laminectomy (uninjured control), mild (0.5 mm displacement), moderate (1.1 mm displacement) and severe (1.3 mm displacement) spinal cord contusions are included to show the degree of axonal sparing that is associated with this hypersensitivity. (Scale bar = 0.5 mm).
<img alt="Figure 5" src="/files/ftp_upload/3247/3247fig5.jpg" /> 
Figure 5. A sample data sheet for up-down von Frey hair testing. The hair value and the result (positive or negative) are recorded for each trial. Indications of supraspinal awareness of the testing are noted. At the end of the test, the responses at each hair value are tallied. The selected threshold value for the example data is circled.

<ol>
	<li>Cruz-Almeida, Y., Felix, E. R., Martinez-Arizala, A., Widerstrom-Noga, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pain+symptom+profiles+in+persons+with+spinal+cord+injury.">Pain symptom profiles in persons with spinal cord injury.</a> Pain Med. 10, 1246-1259 (2009).</li><li>Hulsebosch, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+discovery+to+clinical+trials:+treatment+strategies+for+central+neuropathic+pain+after+spinal+cord+injury.">From discovery to clinical trials: treatment strategies for central neuropathic pain after spinal cord injury.</a> Curr. Pharm. Des. 11, 1411-1420 (2005).</li><li>Kloos, A. D., Fisher, L. C., Detloff, M. R., Hassenzahl, D. L., Basso, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepwise+motor+and+all-or-none+sensory+recovery+is+associated+with+nonlinear+sparing+after+incremental+spinal+cord+injury+in+rats.">Stepwise motor and all-or-none sensory recovery is associated with nonlinear sparing after incremental spinal cord injury in rats.</a> Exp. Neurol. 191, 251-265 (2005).</li><li>Detloff, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+activation+of+microglia+and+pro-inflammatory+cytokines+predict+the+onset+and+severity+of+below-level+neuropathic+pain+after+spinal+cord+injury+in+rats.">Remote activation of microglia and pro-inflammatory cytokines predict the onset and severity of below-level neuropathic pain after spinal cord injury in rats.</a> Exp. Neurol. 212, 337-347 (2008).</li><li>Detloff, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validity+of+acute+and+chronic+tactile+sensory+testing+after+spinal+cord+injury+in+rats.">Validity of acute and chronic tactile sensory testing after spinal cord injury in rats.</a> Exp. Neurol. 225, 366-376 (2010).</li><li>Hutchinson, K. J., Gomez-Pinilla, F., Crowe, M. J., Ying, Z., Basso, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+exercise+paradigms+differentially+improve+sensory+recovery+after+spinal+cord+contusion+in+rats.">Three exercise paradigms differentially improve sensory recovery after spinal cord contusion in rats.</a> Brain. 127, 1403-1414 (2004).</li><li>Carlton, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+and+central+sensitization+in+remote+spinal+cord+regions+contribute+to+central+neuropathic+pain+after+spinal+cord+injury.">Peripheral and central sensitization in remote spinal cord regions contribute to central neuropathic pain after spinal cord injury.</a> Pain. 147, 265-276 (2009).</li><li>Lindsey, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+changes+in+sensory+thresholds+to+mild+tactile+and+cold+stimuli+after+experimental+spinal+cord+injury+in+the+rat.">An analysis of changes in sensory thresholds to mild tactile and cold stimuli after experimental spinal cord injury in the rat.</a> Neurorehabil. Neural Repair. 14, 287-300 (2000).</li><li>Basso, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+testing+after+spinal+cord+injury:+congruities,+complexities,+and+controversies.">Behavioral testing after spinal cord injury: congruities, complexities, and controversies.</a> J. Neurotrauma. 21, 395-404 (2004).</li><li>Langford, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+modulation+of+pain+as+evidence+for+empathy+in+mice.">Social modulation of pain as evidence for empathy in mice.</a> Science. 312, 1967-1970 (2006).</li></ol>

The authors have nothing to disclose.

The VFH testing methods presented here represent two testing paradigms that are sensitive and valid for use in rat SCI models. Importantly, the dorsal VFH test was created for assessment of the recovery of tactile sensation of the hind paws acutely following mid-thoracic SCI when rats are unable to stand on their hindquarters. This testing paradigm may also be used when rats regain the ability to support their weight and for the hind limbs after cervical SCI. 
The two paradigms demonstrated he...

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 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
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 <td>Semmes-Weinstein Monofilaments</td>
 <td>Stoelting Co</td>
 <td>58011</td>
 <td>&nbsp;</td>
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 <td>Fish Breeder Box</td>
 <td>Aquarium Supply Store</td>
 <td>&nbsp;</td>
 <td>Hole drilled in side for cereal delivery</td>
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 <td>Elevated wire mesh (1/4" X 1/4" grid)</td>
 <td>Hardware Store</td>
 <td>&nbsp;</td>
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 <td>Elastic to hold fish breeder box in place</td>
 <td>Craft Store</td>
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 <td>Sugared Cereal</td>
 <td>Grocery Store</td>
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 <td>Surgical Huck Towel 16"x 23"</td>
 <td>Medical Supply Store</td>
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</table>

acute and chronic tactile sensory testing after spinal cord injury in rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

Watch this Scientific Journal Video about Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats at JoVE.com

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

acute-chronic-tactile-sensory-testing-after-spinal-cord-injury

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23.5K Views.  School of Allied Medical Professions, The Ohio State University. We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Video: Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

A Novel Vertebral Stabilization Method for Producing  Contusive Spinal Cord Injury

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing reliable cervical SCI is difficult due to lack of satisfactory methods of vertebral stabilization. The conventional method to stabilize the spine is to suspend the rostral and caudal cervical spine via clamps attached to cervical spinous processes. &#160;However, this method of stabilization fails to prevent tissue yielding during the contusion as the cervical spinal processes are too short to be effectively secured by the clamps (Figure 1). &#160;Here we introduce a new method to completely stabilize the cervical vertebra at the same level of the impact injury. &#160;This method effectively minimizes movement of the spinal column at the site of impact, which greatly improves the production of consistent SCIs. &#160;We provide visual description of the equipment (Figure 2-4), methods, and a step-by-step protocol for the stabilization of the cervical 5 vertebra (C5) of adult rats, to perform laminectomy (Figure 5) and produce a contusive SCI thereafter. &#160;Although we only demonstrate a cervical hemi-contusion using the NYU/MASCIS impactor device, this vertebral stabilization technique can be applied to other regions of the spinal cord, or be adapted to other SCI devices. &#160;Improving spinal cord exposure and fixation through vertebral stabilization may be valuable for producing consistent and reliable injuries to the spinal cord. &#160;This vertebral stabilization method can also be used for stereotactic injections of cells and tracers, and for imaging using two-photon microscopy in various neurobiological studies.

Vertebral stabilization is necessary for minimizing variability, and for producing consistent experimental spinal cord injuries.&#160; Using a customized stabilizing apparatus in conjunction with the NYU/MASCIS impactor device, we have demonstrated here the proper equipment and procedure for generating reproducible hemi-contusive cervical (C5) spinal cord injuries in adult rats.

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing ...

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...

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Subtitles

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats