Name: development of recombinant proteins to treat chronic pain
Uploaded: 2018-04-11T13:30:00
Description: Watch this Scientific Journal Video about Development of Recombinant Proteins to Treat Chronic Pain at JoVE.com

Part of this work has been funded by a Utrecht University Life Sciences grant

All animal experiments were performed in accordance with international guidelines and prior approval from the local experimental ethical committee. Whole blood was obtained from the Mini Donor Service (Mini Donor Dienst, MDD) at the University Medical Center Utrecht (UMCU) in the Netherlands. The MDD has received positive approval from the Medical Ethics Committee of the UMCU (Medisch Ethische Toetscommissie) for the protocol number 07-125/C.
1. Protein Production and Characterization
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<ol>
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This manuscript describes methods for the production and characterization of a recombinant IL4-10 fusion protein, and methods to test its efficacy in inhibiting inflammatory hyperalgesia in mouse models of persistent inflammatory pain. The production and purification of the IL4-10 fusion protein is performed on a small scale. HEK293 cells are selected as an expression system for protein production because they enable post-translational modifications that cannot be achieved in prokaryotic expression systems. Post-translat...

Chronic pain remains one of the most debilitating and under-treated medical problems of the 21st century, affecting &#62;20% of the adult population1,2. However, treatments to provide relief from chronic pain are often ineffective or must be discontinued due to severe side effects3. Importantly, currently available drugs only provide symptomatic relief, but do not significantly modify or cure chronic pain. Although chronic pain appears to be a neurological disorder, evidence suggests involvement of the immune system in chronic pain development4,5. Moreover, immune-based approaches to treat pain are emerging. For example, anti-inflammatory cytokines inhibit pain in several models of chronic pain6,7,8. However, anti-inflammatory cytokines have a short half-life, reducing their potential pain-inhibiting effects. Moreover, anti-inflammatory cytokines work most optimally in concert with each other. To overcome these limitations, we recently fused the anti-inflammatory cytokines interleukin-4 (IL4) and interleukin-10 (IL10) into one molecule. The IL4-10 fusion protein shows superior efficacy in inhibiting chronic inflammatory and neuropathic pain compared to the individual cytokines9. Here we describe how such fusion protein is produced, purified, and how its quality is controlled.
IL4-10 fusion protein is produced in human cells by transient transfection of HEK293-F cells with a pUPE expression vector carrying the cDNA sequence coding the IL4-10 fusion protein. HEK293-F cells are chosen to allow for post-translational modification of the protein, something that does not occur in bacterial expression systems. To optimize glycan capping with sialic acid, cDNA coding beta-galactoside-2, 3-sialyl-transferase is incorporated in the vector as a second transgene. The fusion protein is purified using affinity protein purification of the culture supernatant because it is more powerful than purification by other methods e.g. size-exclusion or ion exchange chromatography10,11. To purify the IL4-10 fusion protein, we used in-house made monoclonal antibodies against IL4. Evaluation of the purity and bioactivity of purified IL4-10 fusion protein is performed as a part of quality control. The purity of produced batches is evaluated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and High Pressure Size Exclusion Chromatography (HP-SEC). The bioactivity of the IL4-10 fusion protein is evaluated by measuring its capacity to inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF&#945;) production in whole blood cultures, and comparing it to the combination of the individual cytokines.
Finally, in order to test the capacity of IL4-10 fusion proteins to inhibit chronic pain, we describe how the fusion protein can be tested as analgesic in widely used mouse models of persistent inflammatory pain12,13,14. Here we describe the methods of an inflammatory pain model. However, it is important to note that other pain models can be used (e.g., neuropathic pain models), depending on the research questions that need to be answered. To assess pain in these models, it is important to use a variety of behavioral measures that include evoked and non-evoked pain measures. Here, we described the methods of assessment for changes in mechanically and thermally evoked behavioral responses. Mechanical sensitivity to innocuous stimuli is assessed using the Von Frey test, whilst thermal sensitivity is assessed using the Hargreaves test. Importantly, non-evoked hyperalgesia/allodynia is measured using the Dynamic Weight Bearing test. These measures are widely accepted as pain measures and yield important information on pain thresholds and potential pain experienced by the animals15,16,17. Other measures to assess non-evoked pain (e.g., stimulus independent), such as the conditioned place preference test, may be valuable18. To assess the potential of the drug to inhibit pain, we performed intrathecal administration of the fusion protein, as with this route of administration less protein dose is needed to reach pain-related areas and avoid systemic (side-) effects19,20.

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			<td height="21" style="height:21px;width:296px;">FreeStyle 293-F cells</td>
			<td style="width:143px;">Invitrogen</td>
			<td style="width:161px;">R790-07</td>
			<td style="width:269px;">Human embryonic kidney cells&#160;</td>
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			<td height="21" style="height:21px;">GIBCO FreeStyle 293 Expression Medium</td>
			<td>Life technologies</td>
			<td>12338018</td>
			<td>Culture medium</td>
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			<td height="21" style="height:21px;">293fectin Reagent</td>
			<td style="width:143px;">Invitrogen</td>
			<td>12347019</td>
			<td>Transfection reagent</td>
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			<td height="21" style="height:21px;">GIBCO Opti-MEM + GlutaMAX</td>
			<td>Life technologies</td>
			<td>51985026</td>
			<td>Reduced Serum Medium for use during cationic lipid transfections&#160;</td>
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			<td height="21" style="height:21px;width:296px;">GIBCO RPMI Medium 1640 (1x)</td>
			<td>Life technologies</td>
			<td style="width:161px;">52400-025</td>
			<td></td>
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			<td height="21" style="height:21px;">CNBr-Activated Sepharose 4B</td>
			<td style="width:143px;">GE Healthcare</td>
			<td>17-0430-01</td>
			<td>pre-activated media for coupling antibodies or other large proteins</td>
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			<td height="21" style="height:21px;width:296px;">Hydrochloric acid fuming 37%&#160;</td>
			<td style="width:143px;">Merck</td>
			<td>1003171000</td>
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			<td height="21" style="height:21px;">Sodium chloride</td>
			<td style="width:143px;">Sigma</td>
			<td>S7653-1kg</td>
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			<td height="21" style="height:21px;width:296px;">Sodium bicarbonate&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:161px;">31437</td>
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			<td height="21" style="height:21px;width:296px;">Trizma hydrochloride</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:161px;">T3253-500G</td>
			<td>TRIS hydrochloride&#160;</td>
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			<td height="21" style="height:21px;">Acetic Acid 100%&#160;&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Merck</td>
			<td style="width:161px;">1.00063.1000&#160;</td>
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			<td height="22" style="height:22px;">Glycin-HCl&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:161px;">G2879&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
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			<td height="21" style="height:21px;">PBS&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:143px;">Pharmacie, UMCU</td>
			<td style="width:161px;"></td>
			<td>Phosphate-Buffered Saline</td>
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			<td height="21" style="height:21px;width:296px;">10X TGS</td>
			<td style="width:143px;">BIO-RAD</td>
			<td style="width:161px;">161-0772</td>
			<td>Tris/Glycine/SDS Buffer for SDS electrophoresis</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Mini-PROTEAN TGX Gels</td>
			<td style="width:143px;">BIO-RAD</td>
			<td style="width:161px;">456-1046</td>
			<td>12% SDS precast gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Trans-Blot Turbo Transfer Pack</td>
			<td style="width:143px;">BIO-RAD</td>
			<td style="width:161px;">170-4157</td>
			<td>Western blot transfer packs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yarra 3u SEC-2000 column</td>
			<td style="width:143px;">Phenomenex</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InstantBlue Protein Stain</td>
			<td style="width:143px;">Expedeon</td>
			<td>ISB1L</td>
			<td>ready to use Coomassie protein stain for polyacrylamide gels</td>
		</tr>
		<tr height="21">
			<td>Human IL-10 DuoSet ELISA</td>
			<td>R&#38;D</td>
			<td>DY217B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>Human TNF&#945; ELISA Set</td>
			<td>Diaclone</td>
			<td>851570020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>BCA Pierce Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carrageenan</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:161px;">22049</td>
			<td>plant mucopolysaccharide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">CFA</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:161px;">F5881</td>
			<td>vaccine adjuvant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Hamilton syringe</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:161px;">20779</td>
			<td>glass syringe</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Animal Enclosure</td>
			<td>IITC Life Science</td>
			<td style="width:161px;">433</td>
			<td style="width:269px;">Animal Enclosure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Von Frey mesh stand</td>
			<td>IITC Life Science</td>
			<td style="width:161px;">410</td>
			<td>Mesh Stand</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">von Frey hairs&#160;</td>
			<td style="width:143px;">Stoelting</td>
			<td>58011</td>
			<td>touch test sensory probes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Plantar Test (Hargreaves Method)</td>
			<td>IITC Life Science</td>
			<td style="width:161px;">390G</td>
			<td>plantar test with heated glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dynamic Weight Bearing test</td>
			<td style="width:143px;">Bioseb</td>
			<td style="width:161px;">BIO-DWB-AUTO-M</td>
			<td>postural deficit test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Econo-Column Columns, 1.5 &#215; 30 cm</td>
			<td style="width:143px;">BIO-RAD</td>
			<td>7371532</td>
			<td>glass chromatography column&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">SnakeSkin Dialysis Tubing&#160;</td>
			<td style="width:143px;">Thermo Scientific</td>
			<td style="width:161px;">88242</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Minisart NML Syringe Filter&#160;</td>
			<td style="width:143px;">Sartorius</td>
			<td style="width:161px;">16555-K</td>
			<td>single use filter unit, 0.45 &#956;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CASY Cell Counter and Analyzer</td>
			<td style="width:143px;">Roche</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

development of recombinant proteins to treat chronic pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.

A representative picture of an SDS-PAGE gel containing different fractions obtained during affinity chromatography purification is shown in Figure 1A. In the load (L) and flow through (FT) fractions, all proteins present in the HEK293 supernatant are observed. No protein is observed in the wash (W) fraction. In the elution (E) fraction, two bands of 35 and 37 kDa are observed corresponding to two different glycoforms of IL4-10 fusion protein ...

Watch this Scientific Journal Video about Development of Recombinant Proteins to Treat Chronic Pain at JoVE.com

Development of Recombinant Proteins to Treat Chronic Pain

In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain.

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

The overall goal of these procedures is to produce a fusion protein of the anti-inflammatory cytokines IL4 and IL10, control its quality, and evaluate its functional activity and analgesic properties in mirroring models of chronic inflammatory pain. This detailed methods aid in developing novel biologics and test their quality and functionality. The advantage of these techniques is that you can produce a sufficient amount of recombinant fusion proteins, which enable its assessment for its analgesic properties in vivo.To evaluate the therapeutic potential of the new developed fusion protein, it is important to have a well-characterized batch and to use a variety of behavioral tests. The implication of these methods is that they provide insight in highly needed new therapies for chronic pain but also extend the potential towards other inflammatory diseases. Generally, scientists new to these methods will struggle to be able to perform all procedures shown here, since expertise in different fields is required.After subculturing 100 milliliters of HEK 293F cells in 500-milliliter Erlenmeyer flasks according to the text protocol, add 6.6 milliliters of DNA transfection reagent mixture to each flask of cells. Culture the cells at 37 degrees Celsius, 8%CO2, and 95%humidity on an orbital shaker rotating at 125 rpm for three days. Then, harvest the culture supernatant containing secreted IL4-10 fusion protein.To carry out protein purification, pass 100 milliliters of 10-fold concentrated cell culture supernatant through a previously prepared, IL4-coupled Sepharose bead column at a flow rate of one milliliter per minute. Use 50 milliliters of PBS to wash the column. Then, apply 12.5 milliliters of 0.1 molar glycine buffer to elute the bound IL4-10 fusion protein into 2.5 milliliter fractions at a flow rate of one milliliter per minute.Next, add 350 microliters of one molar Tris buffer, pH 9, to each fraction to bring the pH to 7. Use dialysis tubing with a 16-millimeter dry diameter and a 3.5K molecular weight cut-off to dialyze the neutralized fractions against two liters of PBS overnight. Then, use disposable filters with 0.45-micrometer pore diameter to filter sterilize the dialyzed fractions.Aliquot the sterile IL4-10 fusion protein solution and store it at negative 80 degrees Celsius. Evaluate the purity of eluted IL4-10 fusion protein by first running a sample on a 12%SDS-PAGE gel and stain the gel with Coomassie blue. Then, carry out high-pressure size-exclusion chromatography by loading 40 microliters of one milligram per milliliter protein onto the column.Determine the concentration of each batch of purified IL4-10 fusion protein using an IL10 ELISA and a BCA protein assay kit according to the manufacturer's protocols. Use RPMI medium to prepare three-fold serial predilutions of the purified IL4-10 fusion protein over a concentration range of five to 1, 215 nanograms per milliliter. Into the wells of a 48-well plate, add 50 microliters of IL4-10 fusion protein, 75 microliters of RPMI medium, 25 microliters of LPS, and 100 microliters of human blood.Incubate the plate at 37 degrees Celsius, 8%CO2, and 95%humidity for 18 hours. Then, use a TNF-alpha ELISA according to the manufacturer's protocol to evaluate the concentration of TNF-alpha in culture supernatants. Maintain prepared carrageenan or CFA solutions in syringes with 30-gauge needles at room temperature for at least 15 minutes before injection.Then, into the thumb of the hind paw of a non-anesthetized mouse, insert the needle approximately 0.5 centimeters, directing it towards the heel and inject 20 microliters of the compound. To carry out pain measurements using the Von Frey test, place an animal into an acrylic cage on a wire-mesh stand and allow the mouse to acclimatize for at least 15 minutes. Apply the Von Frey filaments ranging from 0.02 to four grams perpendicular to the paw surface with sufficient force to cause slight bending against the skin and hold them for approximately three seconds.Measure the paw withdrawal threshold in response to each hair, considering any movement of the paw away from the applied hair as a positive withdrawal response. Place the animals into cages on a pre-warmed glass plate and allow the mice to acclimate for at least 15 minutes prior to measurements. Determine the heat withdrawal latency times by using a Hargreaves apparatus to heat the paw of the animals with a focused visible light beam.Before the dynamic weight-bearing test, measure the body weight of each animal. Place an animal in a floor-instrumented dynamic weight-bearing system assembled onto a cover supporting a camera that will record mouse movements. Allow the animal to acclimate for 0.5 to one minute before recording mouse movements for five minutes.Then, carry out analysis of each video, ensuring that each limb is recognized correctly by the software at the timeframe indicated by the software. Backfill the injectable compounds into a clean 25-microliter glass Hamilton syringe connected to a 27-gauge needle. Place the mouse on the table with its head placed in the nosecone of the apparatus and maintain the mouse sedation with 1.5 to 2%isoflurane with an oxygen flow rate of two liters per minute.Check that the mouse is properly anesthetized by pinching the paw and ensuring that it is nonresponsive. Hold the mouse firmly by spine posterior to the ribs. Position the needle vertically between the L5 and L6 lumbar vertebrae and carefully insert it through the skin into the intervertebral space.Confirm correct targeting by verifying that a tail flick occurred. If only a single paw flick is observed, the needle is likely not placed correctly. Then, slowly inject five microliters of the compound, wait a couple of seconds, and slowly retract the needle.A representative picture of an SDS-PAGE gel containing affinity chromatography fractions is shown here. Two bands of 35 and 37-kilodaltons are observed in the elution fraction corresponding to two different glycoforms of IL4-10 fusion protein. This figure illustrates the results of a potency assay in a whole-blood assay of two different IL4-10 fusion protein batches.A dose-dependent inhibition of LPS-induced TNF-alpha release is observed, showing comparable bioactivity for the two IL4-10 fusion protein batches. In these experiments, persistent inflammatory pain was induced by intraplantar injection of 2%carrageenan. On day six after induction, mice were injected intrathecally with IL4-10 fusion protein.IL4-10 significantly resolved both mechanical and thermal hyperalgesia and reached its maximal effect 24 hours after injection. Here, inflammatory pain was induced by an intraplantar injection of 20 microliters of CFA. Weight-bearing on each paw was measured using the dynamic weight-bearing device.These results show that intraplantar CFA injection reduced weight-bearing of the affected paw. Seven days after intraplantar CFA injection, intrathecal injection of IL4-10 fusion protein was carried out. Two days later, the reduction in weight-bearing of the affected paw was attenuated compared to vehicle-injected mice.Once mastered, using these procedures, three milligram of fusion protein can be obtained from every liter of cultured cells. It is good to keep in mind to choose expression system compatible with future GMP-production of fusion proteins. To test the analgesic potential of this novel fusion protein, other models of chronic pain can be used, like nerve injury or chemotherapy-induced neuropathy.A variety of behavioral tests is shown in this video, but other pain tests suggest conditioned place preference could also be performed. Don't forget that intrathecal injections require well-trained personnel and that the injection volume is rather limited. After watching this video, you should have a good understanding of how to produce a fusion protein and test its efficacy in vivo in various pain models.

development-of-recombinant-proteins-to-treat-chronic-pain

Research

JoVE Journal

Medicine

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

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 ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Chronic Constriction Injury of the Rat's Infraorbital Nerve (IoN-CCI) to Study Trigeminal Neuropathic Pain

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral procedures to study trigeminal neuropathic pain in rats. To meet the specificity of trigeminal neuropathic pain syndromes, the infraorbital nerve (IoN) is subjected to a chronic constriction injury (CCI) by loosely ligating the nerve. An intra-orbital approach is presented here to expose and ligate the IoN in the orbital cavity. After IoN ligation, rats exhibit changes in spontaneous behavior and in response to von Frey hair stimulation that are indicative of persistent pain and mechanical allodynia. Two phases can be defined in the development of the behavioral changes. During the first week following IoN-CCI (phase 1), rats show an increased and asymmetric face grooming activity, i.e., with face wash strokes primarily directed to the nerve-injured IoN territory. A distinction is made between face grooming behavior that is part of a more general body grooming behavior, which remains largely unaffected by IoN-CCI, and face grooming that is neither preceded nor followed by body grooming, which is significantly increased after IoN-CCI. During this period, responsiveness to mechanical stimulation of the IoN territory is reduced. This hyporesponsiveness is abruptly replaced by an extreme hyperresponsiveness whereby even very weak stimulus intensities provoke nocifensive behavior (phase 2). The phenomenological similarities between these behavioral alterations and reported signs of facial pain (i.e., responses to noxious stimulation of the face) suggest the presence of dysesthesia/paresthesia and mechanical allodynia in the ligated IoN territory.

Chronic Constriction Injury of the Rat&#039;s Infraorbital Nerve IoN-CCI to Study Trigeminal Neuropathic Pain

This manuscript describes a rodent model to study trigeminal neuropathic pain. The methods described include surgical procedures to perform a chronic constriction injury of the rat&#8217;s infraorbital nerve and the post-surgical behavioral tests to measure the changes in spontaneous and evoked behavior that are indicative of persistent pain and allodynia.

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral ...

A New Best Practice for Validating Tail Vein Injections in Rat with Near-infrared-Labeled Agents

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part of many experimental procedures where reagents need to be introduced directly into the bloodstream. Unwittingly, the injection can be subcutaneous, possibly altering the scientific outcomes. Utilizing a nanoemulsion-based biological probe with an incorporated near-infrared fluorescent (NIRF) dye, this method offers the capability of imaging a successful tail vein injection in vivo. With the use of a NIRF imager, images are taken before and after the injection of the agent. An acceptable IV injection is then qualitatively or quantitatively determined based on the intensity of the NIRF signal at the site of injection.

Here we present a method to validate tail vein injections in rats by utilizing near-infrared fluorescence imaging data from dyes incorporated into agents or biological probes. The tail is imaged before and after the injection, the fluorescent signal is quantified, and an assessment of the injection quality is made.

Intravenous (IV) administration of agents into the tail vein of rats can be both difficult and inconsistent. Optimizing tail vein injections is a key part ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...

Standardized Tuina Manipulation for Knee Osteoarthritis in Rats

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Author Spotlight: Understanding the Mechanism of Tuina Manipulation in Rats 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...

Standardized Tuina Protocol for Rat DRG Compression Model

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar disc herniation, spinal canal stenosis, and intervertebral foramen stenosis. Although Tuina, a traditional Chinese manual therapy, has shown analgesic effects in clinical practice for the treatment of neuropathic pain, its underlying neurobiological mechanisms remain unclear. Animal models are essential for elucidating the basic principles of Tuina. In this study, we propose a standardized Tuina protocol for rats with compression of the dorsal root ganglion (DRG), which involves inducing DRG compression by inserting a stainless steel rod into the intervertebral foramen, performing Tuina manipulation with specific parameters of location, intensity, and frequency in a controlled environment, and assessing the behavioral and histopathological outcomes of Tuina treatment. This article also discusses the potential clinical implications and limitations of the study and suggests directions for future research on Tuina.

Author Spotlight: Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain  

This article presents a manipulation for treating chronic compression of the dorsal root ganglion in rats using Tuina therapy, along with a method for evaluating its effectiveness based on pain behavior and histopathological results.

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar ...

Description of a Swine Infant Model of Volume-Controlled Hemorrhagic Shock

Hemorrhagic shock is a leading cause of morbidity and mortality in pediatric patients. Interpretation of the clinical indicators validated in adults to guide resuscitation and comparison between different therapies is difficult in children due to the inherent heterogeneity of this population. As a result, compared to adults, appropriate management of pediatric hemorrhagic shock is still not well established. In addition, the scarcity of pediatric patients with hemorrhagic shock precludes the development of clinically relevant studies. For this reason, an experimental pediatric animal model is necessary to study the effects of hemorrhage in children as well as their response to different therapies. We present an infant animal model of volume-controlled hemorrhagic shock in anesthetized young pigs. Hemorrhage is induced by withdrawing a previously calculated blood volume, and the pig is subsequently monitored and resuscitated with different therapies. Here, we describe a precise and highly reproducible model of hemorrhagic shock in immature swine. The model yields hemodynamic data that characterizes compensatory mechanisms that are activated in response to severe hemorrhage.

This article aims to provide researchers with a detailed and accessible guide to set up an infant porcine model of hemorrhagic shock.

Hemorrhagic shock is a leading cause of morbidity and mortality in pediatric patients. Interpretation of the clinical indicators validated in adults to ...

Managing Knee Osteoarthritis Using Ultrasound-Guided Acupotomy

Clinical Effects of Ultrasound-Guided Acupotomy in Knee Osteoarthritis Treatment

The protocol presented here demonstrates the operation method of ultrasound-guided acupotomy for knee osteoarthritis (KOA), including patient recruitment, preoperative preparation, manual operation, and postoperative care. The purpose of this protocol is to relieve pain and improve knee function in patients with KOA. A total of 60 patients with KOA admitted between June 2022 and June 2023 were treated with ultrasound-guided acupotomy. Pathological changes and knee function scores were compared before and after the treatment. After 1 week of treatment, the synovial thickness of the suprapatellar bursae was significantly lesser than before treatment (p &lt; 0.05), the Hospital for Special Surgery Knee Score (HSS) was significantly higher than before treatment (p &lt; 0.05), the Visual analogue scale (VAS) was significantly lower than those of the control group (p &lt; 0.05) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) were significantly lower than those of the control group (p &lt; 0.05). Therefore, ultrasound-guided acupotomy for the treatment of KOA can reduce synovial thickness, relieve pain, improve knee joint function, and have a remarkable curative effect.

Author Spotlight: Minimally Invasive Ultrasound-Guided Acupotomy in Knee Osteoarthritis Treatment

This paper introduces the operation method of ultrasound-guided acupotomy for knee osteoarthritis, which can reduce synovial thickness and improve knee joint function. It has the advantages of precise target, low treatment risk, few complications, and high safety.

The protocol presented here demonstrates the operation method of ultrasound-guided acupotomy for knee osteoarthritis (KOA), including patient recruitment, ...