Peripheral Neuropathy in Mouse Models of Diabetes

Corinne G. Jolivalt1, Katie E. Frizzi1, Lucie Guernsey1, Alex Marquez1, Joseline Ochoa1, Maria Rodriguez1, Nigel A. Calcutt1

1 Department of Pathology, University of California San Diego, La Jolla, California
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.11
Online Posting Date:  September, 2016
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Peripheral neuropathy is a frequent complication of chronic diabetes that most commonly presents as a distal degenerative polyneuropathy with sensory loss. Around 20% to 30% of such patients may also experience neuropathic pain. The underlying pathogenic mechanisms are uncertain, and therapeutic options are limited. Rodent models of diabetes have been used for more than 40 years to study neuropathy and evaluate potential therapies. For much of this period, streptozotocin‚Äźdiabetic rats were the model of choice. The emergence of new technologies that allow relatively cheap and routine manipulations of the mouse genome has prompted increased use of mouse models of diabetes to study neuropathy. In this article, we describe the commonly used mouse models of type 1 and type 2 diabetes, and provide protocols to phenotype the structural, functional, and behavioral indices of peripheral neuropathy, with a particular emphasis on assays pertinent to the human condition. ¬© 2016 by John Wiley & Sons, Inc.

Keywords: allodynia; corneal confocal microscopy; hyperalgesia; hypoalgesia; nerve morphometry; nerve conduction velocity; peripheral neuropathy; skin biopsy; type 1 diabetes; type 2 diabetes

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning: Choosing a Model System
  • Basic Protocol 1: Induction of Diabetes in Mice Using STZ
  • Basic Protocol 2: Measurement of Nerve Conduction Velocity
  • Basic Protocol 3: Measurement of Tactile Allodynia in Diabetic Mice
  • Basic Protocol 4: Morphometry of Myelinated Fibers in Nerve Trunks
  • Basic Protocol 5: Measurement of Paw Thermal Response Latency
  • Basic Protocol 6: Epidermal Innervation
  • Basic Protocol 7: Imaging Corneal Nerves
  • Support Protocol 1: Motor Function Using the Rotarod Test
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Induction of Diabetes in Mice Using STZ

  Materials
  • Mice (see Strategic Planning)
  • Streptozotocin (STZ; Sigma‐Aldrich, cat. no. S0130‐5 G)
  • Normal saline: 0.9% (w/v) NaCl, sterile
  • Glass scintillation vials, 1.5‐ml microcentrifuge tubes, or equivalents
  • 25‐G needles
  • Glucose meter and strips (Lifescan, One Touch Ultra system or equivalent)
  • Additional reagents and equipment for injection of mice (Donovan and Brown, )

Basic Protocol 2: Measurement of Nerve Conduction Velocity

  Materials
  • Mice (see Strategic Planning)
  • Isoflurane
  • Oxygen
  • Povidone‐iodine (Betadine)
  • EZ Anesthesia Versaflex small animal anesthesia system consisting of a water‐circulating heating pad, anesthesia induction chamber, isoflurane regulator, and delivery system (Braintree Scientific, cat. no. EZ‐7150)
  • Sterile surgical instruments
  • Temperature controller set to 37º ± 0.5°C, with fine wire thermistor probe (Yellow Springs Instruments, model 73ATC)
  • Mobile heat lamp connected to the temperature regulator
  • Four platinum‐tipped sub‐dermal needle electrodes (Grass Technologies, cat. no. F‐E2)
  • Laboratory tape
  • PowerLab 4/30 and pre‐amplifier (AD Instruments)
  • Computer running LabChart Pro Software (AD Instruments)

Basic Protocol 3: Measurement of Tactile Allodynia in Diabetic Mice

  Materials
  • Mice (see Strategic Planning)
  • Calibrated von Frey filaments (Kom Kare, Inc.)
  • Electronic von Frey anesthesiometer (IITC Life Sciences Inc.)
  • Testing stand with 2 mm2 mesh surface (self constructed)
  • Glass 250‐ml beakers to act as restraint chambers

Basic Protocol 4: Morphometry of Myelinated Fibers in Nerve Trunks

  Materials
  • Mice (see Strategic Planning)
  • 2.5% glutaraldehyde (see recipe)
  • 0.1 M sodium phosphate buffer, pH 7.3 [1:1 mixture of 0.2 M sodium phosphate buffer, pH 7.3 (see recipe) and distilled H 2O]
  • 2% (w/v) aqueous osmium tetroxide (EMS, cat. no. 19152)
  • 100%, 95%, 70%, 50%, and 30% (v/v) ethanol
  • Propylene oxide (EMS, cat. no. 20411)
  • DDSA (EMS, cat. no. 13701)
  • Araldite 502 resin (EMS, cat. no. 10900)
  • Embed 812 (EMS, cat. no. 14901)
  • DMP‐30 (EMS, cat. no. 13600)
  • Wax or nail polish
  • p‐Phenylenediamine (Sigma, cat. no. P6001)
  • CytoSeal 60 mounting medium (Thermo Fisher Scientific, cat. no. 8310‐4)
  • Dissection tools, including fine forceps
  • Matchstick or wooden applicator
  • 20‐ml scintillation vials (Fisher, cat. no. 450520)
  • Plastic or metal ruler with mm‐markings
  • Plastic or metal ruler with mm markings Double‐edged razor blade (EMS, cat. no. 7200)
  • Tissue rotator (EMS, cat. no. 71780‐10)
  • Plastic disposable beaker of appropriate volume
  • Tissue rotator
  • Vacuum desiccator
  • PELCO 105 flat embedding molds (Ted Pella, cat. no. 105)
  • Labels to identify tissue blocks
  • 10‐ml plastic syringes
  • Oven at 60°C
  • Resin butler block trimmer (EMS, cat. no. 69945‐01)
  • Stereo microscope
  • Single‐edge razor blades (WecPrep, EMS cat. no. 71933‐50)
  • Glass strips (Leica, cat. no. 16840031) and knife maker (Leica, model EM KMR2)
  • Glass knife boats (EMS, cat. no. 71008)
  • Ultramicrotome (Sorvall, model MT‐1)
  • 0.22‐μm nylon filters
  • Non‐coated glass microscope slides (e.g., Superfrost slides; Fisher, cat. no. 12‐550‐123)
  • Glass rods
  • Amber glass bottles
  • Staining jars
  • Glass coverslips (e.g., Fisher, cat. no. 12‐548‐5P)
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, )

Basic Protocol 5: Measurement of Paw Thermal Response Latency

  Materials
  • Mice (see Strategic Planning)
  • Thermal nociception test device/Hargeaves apparatus (UARD) as validated elsewhere (Dirig et al., ).
  • Glass 250‐ml beakers to act as restraint chambers

Basic Protocol 6: Epidermal Innervation

  Materials
  • Mice (see Strategic Planning)
  • 4% buffered paraformaldehyde (see recipe)
  • 0.1 M sodium phosphate buffer, pH 7.3 [1:1 mixture of 0.2 M sodium phosphate buffer, pH 7.3 (see recipe) and distilled H 2O]
  • 100%, 95%, 70%, and 50% ethanol
  • Xylene (Fisher, cat. no. X3S‐4)
  • Paraplast Plus Paraffin (Leica, cat. no. 39602004)
  • 3% hydrogen peroxide in distilled water
  • 1× phosphate‐buffered saline (PBS; see recipe for 5×)
  • Vectastain ABC Kit, Rabbit IgG (Vector Laboratories, cat. no. PK‐4001; includes normal goat serum, secondary antibody, and avidin/biotin complex)
  • Anti‐protein gene product 9.5, rabbit anti‐human, polyclonal IgG (AbD Serotec, cat. no. 7863‐0504)
  • NovaRed Substrate Chromagen Kit (Vector Laboratories, cat. no. SK‐4800)
  • Gill's Hematoxylin (Vector Laboratories, cat. no. H‐3401)
  • CytoSeal 60 mounting medium (Thermo Fisher Scientific, cat. no. 8310‐4)
  • Single‐edge razor blades (WecPrep, EMS cat. no. 71933‐50)
  • Dissection tools
  • Weighing dish or other thin plastic surface
  • 20‐G needle
  • Tissue processing embedding cassettes (EMS, cat. no. 70073‐A)
  • Tissue processor (Leica, model TP1020)
  • Embedding center (Leica, model EG1160)
  • Stainless steel base molds, 15×15×5 mm (EMS, cat. no. 62510‐15)
  • 40ºC water bath
  • Rotary microtome (Leitz, model 1512)
  • Superfrost/Plus slides (Fisher, cat. no. 12‐550‐15)
  • Light microscope
  • 60ºC oven
  • Hydrophobic marker (5 mm PAP Pen, Sigma, cat. no. Z377821)
  • Slide humidifier (StainTray, Simport Plastics, cat. no. M920‐1)
  • Glass coverslips (e.g., Fisher, cat. no. 12‐548‐5P)
  • Lab marker
  • Removable multipurpose stickers
  • Light microscope with digital camera and output to a computer running imaging software (Scion Image freeware or equivalent)
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, )

Basic Protocol 7: Imaging Corneal Nerves

  Materials
  • Mice (see Strategic Planning)
  • Isoflurane
  • Oxygen
  • GenTeal gel (Novartis Pharmaceuticals Corp.)
  • EZ anesthesia Versaflex small animals anesthesia system consisting of a water‐circulating heating pad, anesthesia induction chamber, isoflurane regulator, and delivery system (Braintree Scientific, cat. no. EZ‐7150)
  • Small animal platform (in house‐manufactured)
  • Retina Tomograph 3 with Rostock Cornea Module (Heidelberg Engineering Inc.)
  • Tomocap (Heidelberg Engineering, cat. no. 0220‐001)
  • Rostock Imaging Software (Heidelberg Engineering Inc.)

Support Protocol 1: Motor Function Using the Rotarod Test

  Materials
  • Rotarod device with 1.25 inch diameter mouse rods (Stoelting)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Anderson, N.J., King, M.R., Delbruck, L., and Jolivalt, C.G. 2014. Role of insulin signaling impairment, adiponectin and dyslipidemia in peripheral and central neuropathy in mice. Dis. Model. Mech. 7:625‐633. doi: 10.1242/dmm.015750.
  Beiswenger, K.K., Calcutt, N.A., and Mizisin, A.P. 2008a. Dissociation of thermal hypoalgesia and epidermal denervation in streptozotocin‐diabetic mice. Neurosci. Lett. 442:267‐272. doi: 10.1016/j.neulet.2008.06.079.
  Beiswenger, K.K., Calcutt, N.A., and Mizisin, A.P. 2008b. Epidermal nerve fiber quantification in the assessment of diabetic neuropathy. Acta Histochem. 110:351‐362. doi: 10.1016/j.acthis.2007.12.004.
  Biessels, G.J., Bril, V., Calcutt, N.A., Cameron, N.E., Cotter, M.A., Dobrowsky, R., Feldman, E.L., Fernyhough, P., Jakobsen, J., Malik, R.A., Mizisin, A.P., Oates, P.J., Obrosova, I.G., Pop‐Busui, R., Russell, J.W., Sima, A.A., Stevens, M.J., Schmidt, R.E., Tesfaye, S., Veves, A., Vinik, A.I., Wright, D.E., Yagihashi, S., Yorek, M.A., Ziegler, D., and Zochodne, D.W. 2014. Phenotyping animal models of diabetic neuropathy: A consensus statement of the diabetic neuropathy study group of the EASD (Neurodiab). J. Peripher. Nerv. Syst. 19:77‐87. doi: 10.1111/jns5.12072.
  Bour‐Jordan, H., Thompson, H.L., Giampaolo, J.R., Davini, D., Rosenthal, W., and Bluestone, J.A. 2013. Distinct genetic control of autoimmune neuropathy and diabetes in the non‐obese diabetic background. J. Autoimmun. 45:58‐67. doi: 10.1016/j.jaut.2013.06.005.
  Breyer, M.D., Bottinger, E., Brosius, F.C., 3rd, Coffman, T.M., Harris, R.C., Heilig, C.W., Sharma, K., and Amdcc. 2005. Mouse models of diabetic nephropathy. J. Am. Soc. Nephrol. 16:27‐45. doi: 10.1681/ASN.2004080648.
  Brooks, S.P., Trueman, R.C., and Dunnett, S.B. 2012. Assessment of motor coordination and balance in mice using the rotarod, elevated bridge, and footprint tests. Curr. Protoc. Mouse Biol. 2:37‐53. doi: 10.1002/9780470942390.mo110165.
  Calcutt, N.A. 2004. Modeling diabetic sensory neuropathy in rats. Methods Mol. Med. 99:55‐65.
  Calcutt, N.A., Willars, G.B., and Tomlinson, D.R. 1988. Statil‐sensitive polyol formation in nerve of galactose‐fed mice. Metabolism 37:450‐453. doi: 10.1016/0026‐0495(88)90045‐5.
  Calcutt, N.A., Freshwater, J.D., and Mizisin, A.P. 2004. Prevention of sensory disorders in diabetic Sprague‐Dawley rats by aldose reductase inhibition or treatment with ciliary neurotrophic factor. Diabetologia 47:718‐724. doi: 10.1007/s00125‐004‐1354‐2.
  Calcutt, N.A., Jorge, M.C., Yaksh, T.L., and Chaplan, S.R. 1996. Tactile allodynia and formalin hyperalgesia in streptozotocin‐diabetic rats: Effects of insulin, aldose reductase inhibition and lidocaine. Pain 68:293‐299. doi: 10.1016/S0304‐3959(96)03201‐0.
  Chaplan, S.R., Bach, F.W., Pogrel, J.W., Chung, J.M., and Yaksh, T.L. 1994. Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods 53:55‐63. doi: 10.1016/0165‐0270(94)90144‐9.
  Chen, D.K., Frizzi, K.E., Guernsey, L.S., Ladt, K., Mizisin, A.P., and Calcutt, N.A. 2013. Repeated monitoring of corneal nerves by confocal microscopy as an index of peripheral neuropathy in type‐1 diabetic rodents and the effects of topical insulin. J. Peripher. Nerv. Syst. 18:306‐315. doi: 10.1111/jns5.12044.
  Davidson, E., Coppey, L., Lu, B., Arballo, V., Calcutt, N.A., Gerard, C., and Yorek, M. 2009. The roles of streptozotocin neurotoxicity and neutral endopeptidase in murine experimental diabetic neuropathy. Exp. Diabetes. Res. 2009:431980. doi: 10.1155/2009/431980.
  Dirig, D.M., Salami, A., Rathbun, M.L., Ozaki, G.T., and Yaksh, T.L. 1997. Characterization of variables defining hindpaw withdrawal latency evoked by radiant thermal stimuli. J. Neurosci. Methods 76:183‐191. doi: 10.1016/S0165‐0270(97)00097‐6.
  Donovan, J. and Brown, P. 2006a. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10. doi: 10.1002/0471142735.im0106s73.
  Donovan, J. and Brown, P. 2006b. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4. doi: 10.1002/0471142735.im0108s73.
  Doss, A.L. and Smith, P.G. 2012. Nerve‐Langerhans cell interactions in diabetes and aging. Histol. Histopathol. 27:1589‐1598.
  Gibbons, C.H., Illigens, B.M., Wang, N., and Freeman, R. 2010. Quantification of sudomotor innervation: A comparison of three methods. Muscle Nerve 42:112‐119. doi: 10.1002/mus.21626.
  Grote, C.W., Groover, A.L., Ryals, J.M., Geiger, P.C., Feldman, E.L., and Wright, D.E. 2013. Peripheral nervous system insulin resistance in ob/ob mice. Acta Neuropathol. Commun. 1:15. doi: 10.1186/2051‐5960‐1‐15.
  Guilford, B.L., Ryals, J.M., and Wright, D.E. 2011. Phenotypic changes in diabetic neuropathy induced by a high‐fat diet in diabetic C57BL/6 mice. Exp. Diabetes Res. 2011:848307. doi: 10.1155/2011/848307.
  Guo, G., Kan, M., Martinez, J.A., and Zochodne, D.W. 2011. Local insulin and the rapid regrowth of diabetic epidermal axons. Neurobiol. Dis. 43:414‐421. doi: 10.1016/j.nbd.2011.04.012.
  Jack, M.M., Ryals, J.M., and Wright, D.E. 2011. Characterization of glyoxylase 1 in a streptozotocin‐induced model of diabetes with painful and insensate neuropathy. Diabetologia 54:2174‐2182.
  Johnson, M.S., Ryals, J.M., and Wright, D.E. 2008. Early loss of peptidergic intraepidermal nerve fibers in an STZ‐induced mouse model of insensate diabetic neuropathy. Pain 140:35‐47. doi: 10.1016/j.pain.2008.07.007.
  Kalichman, M.W., Powell, H.C., and Mizisin, A.P. 1998. Reactive, degenerative, and proliferative Schwann cell responses in experimental galactose and human diabetic neuropathy. Acta Neuropathol. 95:47‐56. doi: 10.1007/s004010050764.
  Kennedy, J.M. and Zochodne, D.W. 2005. Experimental diabetic neuropathy with spontaneous recovery: Is there irreparable damage? Diabetes 54:830‐837. doi: 10.2337/diabetes.54.3.830.
  Khan, G.M., Chen, S.R., and Pan, H.L. 2002. Role of primary afferent nerves in allodynia caused by diabetic neuropathy in rats. Neuroscience 114:291‐299. doi: 10.1016/S0306‐4522(02)00372‐X.
  Langford, D.J., Bailey, A.L., Chanda, M.L., Clarke, S.E., Drummond, T.E., Echols, S., Glick, S., Ingrao, J., Klassen‐Ross, T., Lacroix‐Fralish, M.L., Matsumiya, L., Sorge, R.E., Sotocinal, S.G., Tabaka, J.M., Wong, D., van den Maagdenberg, A.M., Ferrari, M.D., Craig, K.D., and Mogil, J.S. 2010. Coding of facial expressions of pain in the laboratory mouse. Nat. Methods. 7:447‐449. doi: 10.1038/nmeth.1455.
  Lauria, G., Hsieh, S.T., Johansson, O., Kennedy, W.R., Leger, J.M., Mellgren, S.I., Nolano, M., Merkies, I.S., Polydefkis, M., Smith, A.G., Sommer, C., Valls‐Sole, J., European Federation of Neurological Societies, and Peripheral Nerve Society. 2010. European Federation of Neurological Societies/Peripheral Nerve Society Guideline on the use of skin biopsy in the diagnosis of small fiber neuropathy. Report of a joint task force of the European Federation of Neurological Societies and the Peripheral Nerve Society. Eur. J. Neurol. 17:903‐912, e944‐909. doi: 10.1111/j.1468‐1331.2010.03023.x.
  Lee‐Kubli, C.A., Mixcoatl‐Zecuatl, T., Jolivalt, C.G., and Calcutt, N.A. 2014. Animal models of diabetes‐induced neuropathic pain. Curr. Top Behav. Neurosci. 20:147‐170. doi: 10.1007/7854_2014_280.
  Leiter, E.H. and Schile, A. 2013. Genetic and pharmacologic models for type 1 diabetes. Curr. Protoc. Mouse. Biol. 3:9‐19. doi: 10.1002/9780470942390.mo120154.
  Lutz, T.A. and Woods, S.C. 2012. Overview of animal models of obesity. Curr. Protoc. Pharmacol. 58:5.61.1‐5.61.18. doi: 10.1002/0471141755.ph0561s58.
  Maier, C., Baron, R., Tolle, T.R., Binder, A., Birbaumer, N., Birklein, F., Gierthmuhlen, J., Flor, H., Geber, C., Huge, V., Krumova, E.K., Landwehrmeyer, G.B., Magerl, W., Maihofner, C., Richter, H., Rolke, R., Scherens, A., Schwarz, A., Sommer, C., Tronnier, V., Uceyler, N., Valet, M., Wasner, G., and Treede, R.D. 2010. Quantitative sensory testing in the German Research Network on Neuropathic Pain (DFNS): Somatosensory abnormalities in 1236 patients with different neuropathic pain syndromes. Pain 150:439‐450. doi: 10.1016/j.pain.2010.05.002.
  Malmberg, A.B., Mizisin, A.P., Calcutt, N.A., von Stein, T., Robbins, W.R., and Bley, K.R. 2004. Reduced heat sensitivity and epidermal nerve fiber immunostaining following single applications of a high‐concentration capsaicin patch. Pain 111:360‐367. doi: 10.1016/j.pain.2004.07.017.
  Mansford, K.R. and Opie, L. 1968. Comparison of metabolic abnormalities in diabetes mellitus induced by streptozotocin or by alloxan. Lancet 1:670‐671. doi: 10.1016/S0140‐6736(68)92103‐X.
  Minett, M.S., Quick, K., and Wood, J.N. 2011. Behavioral measures of pain thresholds. Curr. Protoc. Mouse. Biol. 1:383‐412. doi: 10.1002/9780470942390.mo110116.
  Mounien, L., Marty, N., Tarussio, D., Metref, S., Genoux, D., Preitner, F., Foretz, M., and Thorens, B. 2010. Glut2‐dependent glucose‐sensing controls thermoregulation by enhancing the leptin sensitivity of NPY and POMC neurons. FASEB J. 24:1747‐1758. doi: 10.1096/fj.09‐144923.
  Powell, H.C., Rosoff, J., and Myers, R.R. 1985. Microangiopathy in human diabetic neuropathy. Acta Neuropathol 68:295‐305. doi: 10.1007/BF00690832.
  Quattrini, C., Tavakoli, M., Jeziorska, M., Kallinikos, P., Tesfaye, S., Finnigan, J., Marshall, A., Boulton, A.J., Efron, N., and Malik, R.A. 2007. Surrogate markers of small fiber damage in human diabetic neuropathy. Diabetes 56:2148‐2154. doi: 10.2337/db07‐0285.
  Ramji, N., Toth, C., Kennedy, J., and Zochodne, D.W. 2007. Does diabetes mellitus target motor neurons? Neurobiol. Dis. 26:301‐311. doi: 10.1016/j.nbd.2006.11.016.
  Romanovsky, D., Cruz, N.F., Dienel, G.A., and Dobretsov, M. 2006. Mechanical hyperalgesia correlates with insulin deficiency in normoglycemic streptozotocin‐treated rats. Neurobiol. Dis. 24:384‐394. doi: 10.1016/j.nbd.2006.07.009.
  Schenone, A.E. and Dyck, P.J. 1987. Which endoneurial microvessel histologic measurements are least influenced by vasomotor tone? Brain. Res. 402:151‐154. doi: 10.1016/0006‐8993(87)91058‐4.
  Tomlinson, D.R. and Gardiner, N.J. 2008. Glucose neurotoxicity. Nat. Rev. Neurosci. 9:36‐45. doi: 10.1038/nrn2294.
  Wada, R. and Yagihashi, S. 2004. Nitric oxide generation and poly(ADP ribose) polymerase activation precede beta‐cell death in rats with a single high‐dose injection of streptozotocin. Virchows. Arch. 444:375‐382. doi: 10.1007/s00428‐003‐0967‐z.
  Wahren, J. and Larsson, C. 2015. C‐peptide: New findings and therapeutic possibilities. Diabetes. Res. Clin. Pract. 107:309‐319. doi: 10.1016/j.diabres.2015.01.016.
  Xu, Q.G., Li, X.Q., Kotecha, S.A., Cheng, C., Sun, H.S., and Zochodne, D.W. 2004. Insulin as an in vivo growth factor. Exp. Neurol. 188:43‐51. doi: 10.1016/j.expneurol.2004.03.008.
  Yeomans, D.C. and Proudfit, H.K. 1996. Nociceptive responses to high and low rates of noxious cutaneous heating are mediated by different nociceptors in the rat: Electrophysiological evidence. Pain 68:141‐150. doi: 10.1016/S0304‐3959(96)03177‐6.
  Yeomans, D.C., Pirec, V., and Proudfit, H.K. 1996. Nociceptive responses to high and low rates of noxious cutaneous heating are mediated by different nociceptors in the rat: Behavioral evidence. Pain 68:133‐140. doi: 10.1016/S0304‐3959(96)03176‐4.
  Yorek, M.S., Obrosov, A., Shevalye, H., Lupachyk, S., Harper, M.M., Kardon, R.H., and Yorek, M.A. 2014. Effect of glycemic control on corneal nerves and peripheral neuropathy in streptozotocin‐induced diabetic C57Bl/6 J mice. J Peripher. Nerv. Syst. 19:205‐217. doi: 10.1111/jns.12086.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library