Impaired Nociception in the Diabetic Ins2+/Akita MouseNisha Vastani,1 Franziska Guenther,1,2 Clive Gentry,1 Amazon L. Austin,3 Aileen J. King,3 Stuart Bevan,1 andDavid A. Andersson1

Diabetes 2018;67:1650–1662 | https://doi.org/10.2337/db17-1306

The mechanisms responsible for painful and insensatediabetic neuropathy are not completely understood.Here, we have investigated sensory neuropathy in theIns2+/Akita mouse, a hereditary model of diabetes. Akitamice become diabetic soon after weaning, and we showthat this is accompanied by an impaired mechanical andthermal nociception and a significant loss of intraepi-dermal nerve fibers. Electrophysiological investigationsof skin-nerve preparations identified a reduced rate ofaction potential discharge in Ins2+/Akita mechanonoci-ceptors compared with wild-type littermates, whereasthe function of low-threshold A-fibers was essentiallyintact. Studies of isolated sensory neurons demon-strated a markedly reduced heat responsiveness inIns2+/Akita dorsal root ganglion (DRG) neurons, buta mostly unchanged function of cold-sensitive neurons.Restoration of normal glucose control by islet transplan-tation produced a rapid recovery of nociception, whichoccurred before normoglycemia had been achieved.Islet transplantation also restored Ins2+/Akita intraepider-mal nerve fiber density to the same level as wild-typemice, indicating that restored insulin production can re-verse both sensory and anatomical abnormalities of di-abetic neuropathy in mice. The reduced rate of actionpotential discharge in nociceptive fibers and the im-paired heat responsiveness of Ins2+/Akita DRG neuronssuggest that ionic sensory transduction and transmis-sion mechanisms are modified by diabetes.

Diabetic sensory neuropathy is a major cause of chronicpain and paresthesias, but the most common symptom issensory loss, and insensate neuropathy is a dominant riskfactor for foot ulcers and amputations (1–3). Rodentmodelsof diabetic neuropathy recapitulate many anatomical and

sensory abnormalities observed in patients, and thus appearsuitable for translational mechanistic studies (4,5). Thesensory abnormalities reported from different rodent mod-els differ qualitatively, with diabetic rats commonly display-ing tactile and thermal hypersensitivity, whereas miceusually exhibit loss of sensitivity to mechanical or thermalstimulation similar to that seen in patients (3–5).

Patients with diabetic neuropathy typically display a re-duced tactile sensitivity and a reduced ability to detect skincooling and heating (6–8). A compromised ability to detectstimulation with von Frey filaments and to sense vibrationare thus important and simple diagnostic tools for earlysigns of diabetic neuropathy (9,10). In addition to the lossof sensation and the development of pain and paresthe-sias, diabetic neuropathy is characterized by reduced nerveconduction velocity, distal fiber loss and reduced axondiameters (11,12). The relative importance of abnormal-ities in sensory transduction, conduction, and anatomicalstructure for painful or insensate neuropathy in vivo iscurrently unclear, but the presence of sensory abnormal-ities cannot be used to discriminate between patientswith painless and painful diabetic neuropathy (12).

Here, we present a detailed characterization of sensoryneuropathy in the Ins2+/Akita mouse using behavioral,cellular, and neurophysiological approaches. Mice thatare heterozygous for the Ins2C96Y (Ins2+/Akita) mutationdevelop b-cell endoplasmic reticulum stress, produce littleinsulin, and become diabetic soon after weaning (13,14).The phenotype is more serious in male mice than in fe-male mice (13,14). Since the Ins2C96Ymutation leads to thespontaneous development of diabetes, the Akita strainprovides a convenient translational model suitable forinvestigations of diabetic complications and islet trans-plantations (15). Relatively little information is available

1Wolfson Centre for Age-Related Diseases, Institute of Psychiatry, Psychology &Neuroscience, King’s College London, London, U.K.2Institut für Physiologie und Pathophysiologie, Universität Erlangen-Nürnberg,Erlangen, Germany3Diabetes & Nutritional Sciences Division, King’s College London, London, U.K.

Corresponding author: David A. Andersson, david.andersson@kcl.ac.uk.

Received 27 October 2017 and accepted 18 May 2018.

N.V. and F.G. are joint first authors.

© 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

1650 Diabetes Volume 67, August 2018

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about the sensory phenotype of Ins2+/Akita mice, whereasautonomic neuropathy has been examined in more de-tail previously (16). The results reported here show thatIns2+/Akita mice rapidly develop impaired mechanosensa-tion after the onset of hyperglycemia, later followed byimpaired thermal (hot and cold) nociception. Investigationsof skin-saphenous nerve preparations demonstrate a reducedrate of action potential discharge in mechanosensitive A- andC-nociceptors at high stimulus intensities. The reduced be-havioral sensitivity to noxious heat was associated witha reduced proportion of heat-sensitive dorsal root gan-glion (DRG) neurons in Ins2+/Akitamice, as well as a reduced[Ca2+]i response amplitude in heat-sensitive neurons.

RESEARCH DESIGN AND METHODS

Mice and Behavioral TestsBehavioral experiments were carried out according tothe U.K. Home Office Animal Procedures (1986) Act. Allprocedures were approved by the King’s College LondonAnimal Welfare and Ethical Review Body and were con-ducted under the U.K. Home Office Project License PPL70/7510. Ins2+/Akita mice were obtained from The JacksonLaboratory (stock #003548, mouse genome informatics#1857572; Bar Harbor, ME) and maintained on theC57BL/6J strain. Blood glucose was monitored routinelyby Contour XT blood glucose meter (Bayer Healthcare,Reading, U.K.) or Stat Strip Xpress meter and strips (NovaBiomedical, Runcorn, U.K.).

The Randall-Selitto paw-pressure test was performedusing an Analgesy-Meter (Ugo Basile, Gemonio, Italy). Micewere kept in their holding cages to acclimatize (10–15 min)to the experimental room. The experimenter lightly re-strained the mouse and applied a constantly increasingpressure stimulus to the dorsal surface of the hind pawusing a blunt conical probe. The nociceptive threshold wasdefined as the force in grams at which the mouse withdrewits paw. A force cutoff value of 150 g was used to avoidtissue injury.

Tactile sensitivity was assessed using von Frey filaments(0.008–2 g) according to the up-down method of Chaplanet al. (17). Animals were placed in a Perspex chamber witha metal grid floor allowing access to their plantar surfaceand were allowed to acclimatize prior to the start of theexperiment. The von Frey hairs were applied to the plan-tar surface of the hind paw with enough force to allowthe filament to bend and were held static for ;2–3 s. Thestimulus was repeated up to five times at intervals ofseveral seconds. The stimulus interval was adapted to allowfor the resolution of any behavioral responses to previousstimuli. A positive response was noted if the paw wassharply withdrawn or if the mouse flinched upon removalof the hair. Any movement of the mouse, such as walkingor grooming, was deemed an unclear response, and in suchcases the stimulus was repeated. If no response was noted,a higher force hair was tested and the filament produc-ing a positive response was recorded as the threshold.

Thermal nociception was examined by lightly restrain-ing the animal and placing one of the hind paws onto a coldor hot plate maintained at 10°C or 50°C (18,19). The pawwithdrawal or escape latency was measured using 30 s asa cutoff to avoid tissue injury.

Skin-Nerve PreparationsSingle-fiber recordings were carried out on preparationsfrom 18- to 20-week-old adult male littermate Ins2+/+ andIns2+/Akita mice. The saphenous nerve and hind-paw hairyskin were dissected free and immediately placed in warm(32°C) oxygenated synthetic interstitial fluid (SIF) con-taining the following (in mmol/L): 108 NaCl, 3.5 KCl, 0.7MgSO4, 26.2 NaHCO3, 1.65 NaH2PO4, 9.6 Na-gluconate,5.55 glucose, and 1.53 CaCl2, buffered to pH 7.4 bybubbling carbogen (95% O2 and 5% CO2). The skin waspinned down, corium side up, and the saphenous nerve wasplaced on a mirrored platform covered in paraffin oil ina separate chamber containing an SIF-paraffin oil interface.The nerve was desheathed and teased into thin filaments,which were placed onto a gold electrode. Electrical signalswere recorded using the DAM80 differential amplifier(World Precision Instruments), band-pass filtered at300Hz and 10 kHz, and digitized and stored on a computerusing the Micro1401 data acquisition unit and SPIKE2version 8 software (Cambridge Electronic Design). Actionpotentials were also visualized using an oscilloscope (Gould420 series).

Receptive fields of mechanosensitive primary afferentslocated in the hairy skin of the hind paw and lower hindlimb were identified using a blunt glass rod and thereafterstimulated with brief electrical pulses (1 ms; DS2; Digi-timer) to determine the conduction velocity of the fiber.Fibers with conduction velocity,1.2 m/s were classified asC fibers, those between 1.2 and 10 m/s as Ad fibers, andthose with conduction velocity .10 m/s as Ab fibers (20).

The mechanical threshold of single fibers was assessedby identifying the minimum force (delivered as a 2-s stepby a computer-driven, feedback-controlled stimulator) re-quired to evoke two action potentials (21). Adaptationproperties were characterized by applying 15 s of sus-tained mechanical force of 0.5, 1, 2, 4, 5, 10, 15, and 20 gwith 2-min interstimulus intervals to reduce tachyphy-laxis. The encoding properties of mechanosensitive Adand C fibers were examined by applying ramp-shaped me-chanical stimuli.

The thermal sensitivity of some C fibers was investi-gated. A metal ring was used to isolate the receptive fieldand a 60-s cold stimulus of ;31–4°C was applied bysuperfusing precooled SIF. Thereafter, a 20-s heat stimulusof ;32–48°C was applied by prewarming the SIF througha heated element. The rate of temperature increase wasdetermined using a custom-made feedback-controlled de-vice. For both the cold and heat stimuli, peristaltic pumpswere used to control the SIF flow rate. A digital temper-ature probe (GIA 2000; Greisinger) was placed inside thering to record the temperature at the skin. A thermally

diabetes.diabetesjournals.org Vastani and Associates 1651

elicited response was considered if a minimum of twoaction potentials occurred and the temperature at whichthe second action potential fired was recorded as the thermalthreshold.

Data were analyzed offline using the SPIKE2 software(Cambridge Electronic Design). Action potentials werediscriminated using templates based on their amplitudeand waveform.

DRG NeuronsDRG neurons were dissociated enzymatically (using colla-genase and trypsin) from ganglia dissected from 12-week-old (one of each phenotype) or 20-week-old (two to threeof each genotype) male littermate Ins2+/+ and Ins2+/Akita

mice using methods described previously (22). Isolatedneurons were plated on poly-D-lysine–coated coverslips andmaintained at 37°C in 95% air and 5% CO2. Wild-type(WT; Ins2+/+) neurons were maintained in MEM AQ con-taining 5.6 mmol/L glucose (Sigma-Aldrich, Poole, U.K.)and Ins2+/Akita neurons in DMEM GlutaMAX containing25 mmol/L glucose (Thermo Fisher Scientific, Paisley, U.K.),each supplemented with 10% FBS, 100 units/mL penicillin,100 mg/mL streptomycin, 10 mmol/L cytosine arabinoside,and 50 ng/mL nerve growth factor (Promega, Southampton,U.K.) for up to 24 h before experimentation. The serumcontent of insulin makes the final concentration in medium4 pmol/L, which is far below that required to supporttrophic effects on isolated DRG neurons (23).

Intracellular [Ca2+]i MeasurementsDRG neurons were loaded with 2.5 mmol/L Fura-2 AM(Invitrogen) in the presence of 1 mmol/L probenecid and0.01% pluronic acid for ;1 h at 37°C. In all experiments,the cells were constantly superfused with physiologicalsaline solution containing the following (in mmol/L):140 NaCl, 5 KCl, 10 glucose, 10 HEPES, 2 CaCl2, and1 MgCl2, pH 7.4 (NaOH). Fura-2 was excited at wave-lengths of 340 and 380 nm, and light emission wasmeasured at wavelengths .520 nm. Solutions were ap-plied through a tube placed in close proximity to the cellsand ratio images were captured every 2 s. Data wereanalyzed using ImageMaster software (Media Cybernetics,Rockville, MD) to obtain emission intensity ratios(R(340/380)) in individual cells.

Intraepidermal Nerve FibersHind paw skin was dissected and fixed in 4% paraformal-dehyde in PBS and kept at room temperature until em-bedded in paraffin wax. The 8-mm-thick sections were cutand stained with rabbit polyclonal anti-PGP9.5 (UltracloneLimited, Isle of Wight, U.K.) overnight at 4°C. Stainingwas visualized using a biotinylated goat anti-rabbit IgG(Vector Laboratories), followed by Streptavidin-Alexa Fluor568 (Thermo Fisher Scientific). Skin sections were addi-tionally stained with DAPI to visualize nuclei. The highdensity of nuclei in the epidermis provides a clear in-dication of the location of the basement membrane. Wehave adapted the general method described for human

tissue (24,25) to suit thin sections prepared from mouseskin (26,27). The number of intraepidermal nerve fibers(IENFs) extending intact from the basement membrane permillimeter was quantified using a Zeiss Apotome Mi-croscope. The observer was blinded to the identity of theanimal.

Islet Isolation and TransplantationIslets were isolated from 10- to 12-week-old C57BL/6Jmice (Charles River, Margate, U.K.) using collagenase di-gestion (1 mg/mL, type XI; Sigma-Aldrich, Gillingham,U.K.) and density gradient separation (Histopaque-1077;Sigma Aldrich), as previously described (28). Islets werewashed in culture medium (RPMI 1640 medium supple-mented with 10% FBS and 1% Penicillin/Streptomycin) anddivided into groups of 500 for same-day transplantation.

Male 14- to 20-week-old Ins2+/Akita mice were trans-planted with 500 freshly isolated islets into the renalsubcapsular space. Mice were anesthetized with 1–5%isoflurane and 95% oxygen (1 L/min). The kidney wasexposed by a lumbar incision, and a small perforation inthe kidney capsule was made using a 23-gauge needle.Islets were pelleted in PE50 tubing before being injectedunder the renal capsule using a Hamilton syringe (HamiltonRobotics, Reno, NV). The capsule was then cauterized andthe mouse peritoneum and skin were sutured. Mice weregiven carprofen (4 mg/kg s.c.; Caprieve; Norbrook Labora-tories Limited,Newry,Northern Ireland, U.K.) and bupivacaine(0.5% solution, 2 mg/kg s.c.; Marcaine; Aspen Medical,London, U.K.) for postoperative analgesia.

Data AnalysisQuantitative data are presented as the individual raw datapoints or the mean 6 SEM, and the number of observa-tions (animals or cells indicated by n). Data were analyzedby t test, Mann-Whitney U test, or ANOVA followed byTukey post hoc test as appropriate. Categorical data wereanalyzed by Fisher exact test. Boxplots show the medianand interquartile range, with whiskers indicating the 5thand 95th percentile. Statistical analysis was performedusing Statistica or IBM SPSS (version 20 or higher). Differ-ences were considered to be significant at P , 0.05.

Drugs and ChemicalsIcilin was purchased from Tocris Bioscience (Bristol, U.K.),salts and all other reagents from Sigma-Aldrich, unlessstated otherwise.

RESULTS

Diabetes in Ins2+/Akita MouseIns2+/Akita mice developed hyperglycemia soon after wean-ing and became diabetic at ;4–5 weeks of age (Fig. 1A).Hyperglycemia progressively worsened in male Ins2+/Akita

mice, and at ;12 weeks of age, their blood glucose valuesreached or exceeded the upper limit of most blood glucosemeters (;33mmol/L). In contrast, blood glucose peaked at;6 weeks of age in female Ins2+/Akita mice and thereafterdeclined toward normoglycemia. Male, but not female,

1652 Sensory Neuropathy in the Ins2+/Akita Mouse Diabetes Volume 67, August 2018

Ins2+/Akita mice gained body weight at a reduced ratecompared with WT littermates (Fig. 1B).

Nociception in the Ins2+/Akita MouseWe assessed the behavioral sensitivity of male and femaleWT and Ins2+/Akitamice to noxious mechanical and thermalstimulation, starting soon after weaning and continuinguntil ;20 weeks of age. Mechanical nociception wasassessed using the Randall-Selitto paw-pressure test andpunctate stimulation with von Frey filaments, and thesensitivity to noxious thermal stimulation was determinedusing a modified cold- and hot-plate assay (18,19). Bothmale and female Ins2+/Akita mice developed a reducedsensitivity in the paw pressure test, almost immediatelyafter the onset of hyperglycemia (Fig. 1C). Furthermore,female Ins2+/Akita mice progressively recovered from me-chanical insensitivity from ;10–12 weeks, when theirblood glucose concentration started to decline. From14 weeks of age, female Ins2+/Akita mice were no longer

distinguishable from WT littermates in the paw-pressuretest, suggesting that the glycemic profile has a dynamicinfluence on mechanical nociception in mice (Fig. 1C).The paw withdrawal threshold in response to stimulationwith calibrated von Frey filaments did not differ betweenIns2+/Akita and WT littermates, apart from a transientlyreduced sensitivity in male Ins2+/Akitamice at;10 weeks ofage (Fig. 1D).

The paw withdrawal latency of male Ins2+/Akita mice inthe cold-plate (10°C) and hot-plate (50°C) tests increasedat ;9–10 weeks of age and thereafter remained ele-vated until at least 19–20 weeks of age (Fig. 1E and F).In contrast, female Ins2+/Akita mice remained indistin-guishable fromWTmice in both thermal tests, apart froma transiently reduced cold sensitivity at ;10 weeks of age(Fig. 1E and F). The paw pressure thus appears to bea particularly sensitive parameter for assessing sensoryabnormalities in diabetic mice, and our results indicatethat the degree of hyperglycemia experienced by femaleIns2+/Akita mice is not sufficient to influence the behavioralsensitivity to noxious hot or cold stimulation.

Low-Threshold MechanoreceptorsThe conduction velocities and the mechanical responsethresholds of rapidly adapting (RA), slowly adapting (SA)Ab, and Ad down hair (DH) fibers were unchanged inpreparations from 18- to 20-week-old male diabeticIns2+/Akita mice compared with fibers in age-matchedmale WT littermates (Table 1). Furthermore, RA andDH fibers from diabetic Ins2+/Akita mice displayed an un-changed action potential firing pattern to sustained forceacross the range of forces tested (0.5–20 g) compared withfibers in preparations from WT littermates (Fig. 2A, B, E,and F). We did note a significantly reduced firing rate inIns2+/Akita SA fibers in response to the lowest force (0.5 g;P = 0.01, Mann-Whitney U test) (Fig. 2C). This observationsuggests that SA fibers in Ins2+/Akita mice display an accel-erated adaptation at low stimulus strengths, compared withWT mice (Fig. 2D).

Ad- and C-MechanoreceptorsThe conduction velocities of Ins2+/Akita nociceptive Ad-mechanoreceptors (AMs) (29) were unchanged comparedwith WT fibers in preparations from 20-week-old malemice. Unmyelinated C-mechanoreceptors (CMs) (30) fromIns2+/Akita mice, however, displayed a reduced conductionvelocity compared with WT fibers (P = 0.007, Mann-Whitney U test) (Table 1), as previously noted in diabeticrats (31). The response threshold in AM fibers fromdiabetic mice was modestly increased (Table 1), but themost striking differences between the response propertiesof nociceptors from Ins2+/Akita and WT mice were revealedby suprathreshold stimulation. WT AM fibers faithfullyencoded mechanical force with a linear increase in dis-charge rate. In contrast, fibers from Ins2+/Akita mice failedto encode stimuli exceeding 10 g (Fig. 3A) and displayeda significantly reduced impulse rate in response to the

Figure 1—Diabetes and nociception in Ins2+/Akita mice. Blood glu-cose (A) and body weight (B) were monitored weekly in male andfemale Ins2+/Akita mice and WT littermates. Mechanical nociceptionwas monitored using the paw-pressure test (C ) and punctate stim-ulation with von Frey filaments (D). Thermal nociception was exam-ined using cold-plate (10°C, E) and hot-plate (50°C, F) tests. *P ,0.05, **P , 0.01, and ***P , 0.001, compared with WT mice of thesame sex, ANOVA followed by Tukey test. N = 3–20 (3 for somemeasurements at 3–4 and ,18 weeks of age).

diabetes.diabetesjournals.org Vastani and Associates 1653

highest mechanical forces (Fig. 3A and D). The temporalresponse profiles evoked by force steps demonstrate thatIns2+/Akita AM fibers failed to reach the maximal initialimpulse rate typical of WT fibers (Fig. 3B and C). Themechanical encoding properties of AMs were examinedfurther using ramp-shaped stimuli. Diabetic AM fibersdisplayed marginally (but not significantly) reduced aver-age firing frequencies throughout the range of ramp forcesused (Fig. 3E and F), an observation that may be consis-tent with a failure to achieve the initial peak firing rate(Fig. 3C), but a retained ability to encode force in responseto continuously increasing ramp stimulation.

Stimulation with force steps produced similar impulsedischarge rates in CM fibers from WT and Ins2+/Akita mice(Fig. 4A and B), but the responses to the maximal rampforce were significantly reduced in Ins2+/Akita CM fibers(Fig. 4C). Although Ins2+/Akita CM fibers encoded the in-tensity of a ramp force stimulus, they appeared to do so ata somewhat lower rate than CM fibers in WT preparations(Fig. 4D and E).

Heat and Cold Sensitivity of Ins2+/Akita CMsSince diabetic mice displayed behavioral insensitivity tonoxious heat and cold, we investigated whether the ther-mal sensitivity of CMs was altered in Ins2+/Akita mice. Theproportion of C-fiber nociceptors that responded to cold(WT: 48%, n = 14 of 29 vs. Ins2+/Akita: 48%, n = 12 of 25; P =0.98, Pearson x2 test) or heat (WT: 51%, n = 15 of 29 vs.Ins2+/Akita: 45%, n = 9 of 20; P = 0.64, Pearson x2 test) wasunchanged in Ins2+/Akita mice compared with WT litter-mates. Cold-sensitive nociceptors (CMC fibers) from di-abetic mice exhibited a marginally, but not significantly,reduced temperature threshold for activation (Ins2+/Akita

18.2 6 2°C; WT 21.4 6 1.7°C; P = 0.25, Mann-WhitneyU test) (Fig. 5A and B). Cold stimulation evoked a signif-icantly reduced number of action potentials in CMC fibersfrom Ins2+/Akita mice compared with WT mice (Ins2+/Akita

18 6 4 action potentials; WT 37 6 7 action potentials,respectively; P = 0.018, Mann-Whitney U test) (Fig. 5C–E).Similarly, heat-sensitive nociceptors (CMH fibers) fromdiabetic mice displayed slightly, but not significantly,higher thresholds for heat activation (Ins2+/Akita 40.4 61.2°C; WT 38.4 6 1.0°C; P = 0.14, Mann-Whitney U test)(Fig. 5F and G) and a reduced number of action potentials

in CMH fibers from Ins2+/Akita compared with WT mice(Ins2+/Akita 16 6 5 action potentials; WT 25 6 5 actionpotentials, respectively; P = 0.18, Mann-Whitney U test)(Fig. 5H–J).

Heat and Cold Sensitivity of Ins2+/Akita DRG NeuronsWe used [Ca2+]i imaging to determine whether the behav-ioral insensitivity of Ins2+/Akita mice to noxious heat andcold was associated with a compromised thermal sensitiv-ity of isolated DRG neurons (Fig. 6A and B). These experi-ments were performed on neurons isolated from 12- to20-week-old male WT and Ins2+/Akita mice. Stimulationwith heat ramps (from 25 to 48°C) evoked [Ca2+]iresponses in a significantly reduced proportion of DRGneurons from Ins2+/Akita mice compared with neuronsisolated from WT mice (Ins2+/Akita mice: 19.3%, 428 of2,222, n = 4 animals; WT mice: 28.5%, 300 of 1,054, n = 3animals; P , 0.001, Fisher exact test). Our measurementsrevealed a modestly, but significantly, reduced tempera-ture threshold for heat activation in neurons from diabeticmice (Ins2+/Akita 41.5 6 0.2°C; WT 42.9 6 0.2°C) (Fig. 6C)(P , 0.001, t test). The heat-evoked [Ca2+]i responseamplitude was reduced by 40% in neurons from Ins2+/Akita

mice compared with WT mice (Ins2+/Akita 0.77 6 0.02R(340/380); WT mice 1.2 6 0.04 R(340/380) (Fig. 6D) (P ,0.001, t test). These results strongly suggest that animpaired neuronal transduction of noxious heat maycontribute to the behavioral insensitivity observedin vivo. The proportion of neurons responding to thetransient receptor potential vanilloid 1 (TRPV1) agonist1 mmol/L capsaicin (Ins2+/Akita neurons: 44.3%, 759 of1,715 neurons; WT neurons: 42.4%, 447 of 1,054; P. 0.3,Fisher exact test) and the response amplitude evoked bycapsaicin were indistinguishable in cultures from WT andIns2+/Akita mice, indicating that the reduced heat respon-siveness of Ins2+/Akita DRG neurons is not likely due to analtered expression of the noxious heat transducer TRPV1.

In contrast to the reduced heat sensitivity of Ins2+/Akita

DRG neurons, cold ramps (35–10°C) elicited indistinguish-able [Ca2+]i responses in neurons isolated from mice ofthe two genotypes. Cooling stimulated [Ca2+]i responsesin a small proportion of DRG neurons from 20-week-oldIns2+/Akita (2.4%, 55 of 2,318) and WT littermates (2.5%,40 of 1,604). Cold-evoked [Ca2+]i response amplitudes did

Table 1—Sensory nerve fiber conduction velocity and mechanical thresholds in male 20-week-old WT and diabetic Ins2+/Akita

mice

Fiber typeWT conductionvelocity (m/s)

Ins2+/Akita

conductionvelocity (m/s) P values

WT mechanicalthreshold (g)

Ins2+/Akita mechanicalthreshold (g) P values

Ab RA 14.3 6 1.1 (20) 13.4 6 0.7 (10) 0.71 0.5 6 0.2 (14) 0.7 6 0.3 (10) 0.84

Ab SA 15.8 6 1.0 (21) 15.5 6 2.0 (9) 0.44 0.4 6 0.1 (12) 0.5 6 0.1 (9) 0.23

Ad DH 7.5 6 0.3 (16) 6.4 6 0.6 (11) 0.27 0.2 6 0.04 (12) 0.2 6 0.1 (11) 0.34

AM 5.9 6 0.4 (38) 6.8 6 1.0 (12) 0.47 2.3 6 0.3 (32) 2.9 6 0.3 (12) 0.037

CM 0.57 6 0.02 (45) 0.49 6 0.02 (28) 0.007 2.3 6 0.2 (35) 3.0 6 0.5 (24) 0.9

Values are the mean 6 SEM (n). P values are from Mann-Whitney U tests.

1654 Sensory Neuropathy in the Ins2+/Akita Mouse Diabetes Volume 67, August 2018

not differ between neurons of the two genotypes (Fig. 6F),whereas the cold activation temperature was marginally,but significantly, higher in Ins2+/Akita neurons (24.5 60.7°C) compared with WT neurons (22.3 6 0.9°C) (Fig.6E). These results indicate that the behavioral insensitivityto noxious cold observed in diabetic Ins2+/Akita mice is notassociated with compromised cellular cold transductionmechanisms.

Reduced Cutaneous Innervation in Diabetic Ins2+/Akita

MiceDiabetic neuropathy is accompanied by a loss of IENFs inpatients and in animal models of diabetes, a feature that is

frequently used as a diagnostic tool in various small-fiberneuropathies (25,32,33). Plantar skin sections preparedfrom male 20-week-old diabetic Ins2+/Akita mice andWT littermates were immunohistochemically labeled forthe expression of the pan-neuronal marker PGP9.5 (Fig.7A). We measured the IENF density by counting thenumber of nerve fibers (in mm21) that extended intactfrom the epidermal/dermal junction into the epidermis, inthin (8 mm) paraformaldehyde-fixed paraffin-embeddedsections of plantar skin (24,25,32). Our results revealed asignificant loss of IENFs in diabeticmice. Comparedwith pawskin from WT littermates, the density of PGP9.5-positiveepidermal fibers was reduced by about a third in diabeticIns2+/Akita mice (Fig. 7B).

Islet Transplantation Rapidly Reversed Symptoms ofSensory NeuropathyFinally, we examined whether the impaired nociceptionand loss of IENFs in Ins2+/Akita mice could be reversed byimproved glycemic control. A group of diabetic Ins2+/Akita

mice underwent the transplantation of pancreatic isletsobtained from healthy C57BL/6J mice (500 islets underthe kidney capsule). After transplantation, blood glucoselevels declined steadily during the first few days aftertransplantation, followed by a slower phase of decline,and at 3 weeks after islet implantation all mice had glucoselevels ,15 mmol/L (Fig. 7C). Intriguingly, nociceptionimproved rapidly and significantly after transplantation(Fig. 7D and E). The behavioral sensitivity to noxiousmechanical and cold stimulation recovered to the samelevel as in naive WT mice within a few days of trans-plantation, and thus occurred before normoglycemia wasestablished. The IENF density in paw skin from trans-planted mice had increased significantly at the end of theexperiment (5–6 weeks after transplantation) comparedwith Ins2+/Akita mice that had not received an islet trans-plant. The IENF density in transplanted mice was indis-tinguishable from that in healthy WT mice (Fig. 7B).

DISCUSSION

Here, we present a detailed characterization of sensoryneuropathy in the Ins2+/Akita mouse, and demonstrate thatdiabetes leads to an impaired mechanical and thermal(hot and cold) nociception, which is associated witha marked loss of IENFs. We noted a rapid loss of me-chanical nociception at the onset of diabetes, whichwas followed by a reduced sensitivity to noxious coldand heat 2–3weeks later. In contrast, we did not observe analtered sensitivity to stimulation with von Frey filaments.We used manual stimulation with calibrated von Freyfilaments according to the up-down method of Chaplanet al. (17), rather than the widely used automatedvon Frey assay (5), which uses stimuli of higher forceintensity and generates higher paw withdrawal thresholds.Our behavioral and electrophysiological studies suggestthat mechanonociceptors are particularly sensitive to di-abetes, whereas low-threshold A-fibers are functionally

Figure 2—Diabetes has a limited impact on low-threshold mecha-noreceptors. The number of action potentials elicited per second bya 15-s force step in skin-nerve preparations from 20-week-old maleWT and Ins2+/Akita mice is shown for RA, SA, and DH fibers. RA (A)and DH (E) fibers from Ins2+/Akita and WT littermates respondedsimilarly in response to all forces. The pattern of temporal responseto sustained stimuli was indistinguishable betweenWT and Ins2+/Akita

RA (B) and DH (F ) fibers, with high rates of action potentials at theforce onset and offset. The mean response to 0.5 g constant force isshown. C: Diabetic SA fibers responded significantly less to thelowest force of 0.5 g (P = 0.01, Mann-Whitney U test).D: Diabetic SAfibers showed a reduced firing rate after the first second to a 0.5-gsustained force. **P, 0.01, compared with SA fibers fromWT mice,Mann-Whitney U test.

diabetes.diabetesjournals.org Vastani and Associates 1655

more resilient. Intriguingly, the loss of mechanical noci-ception appears to be a dynamic process, with rapid onsetafter the establishment of hyperglycemia, and a similarly

rapid recovery following islet transplantation. In addi-tion, the recovery of female Ins2+/Akita mice from hyper-glycemia was accompanied by a normalized sensitivity in

Figure 3—AMs are impaired in diabetic Ins2+/Akita mice. A: Ins2+/Akita AM fibers displayed a reduced rate of action potential discharge inresponse to 15-s applications of 15 and 20 g of force, comparedwithWT fibers (P = 0.008 for 15 g, P = 0.0012 for 20 g, Mann-WhitneyU test).B: Ins2+/Akita AM fibers display a reduced rate of firing in response to application of 20 g of force, with a particularly marked reduction duringthe first few seconds of the stimulus. C: WT AM fibers attained significantly higher maximal firing rates in response to applications of both15 and 20 g (P = 0.002 for 15 g andP = 0.005 for 20 g,Mann-WhitneyU test) comparedwith diabetic AM fibers.D: Example traces of Ins2+/Akita

andWT AM fibers demonstrating the markedly reduced response rate in diabetic AM fibers. E: Ins2+/Akita andWT AM fiber responses evokedby increasing force ramps. F: The temporal pattern of impulse discharge rates evoked by 20-g force ramps in Ins2+/Akita and WT AM fibers.**P , 0.01, compared with WT, Mann-Whitney U test. All fibers were recorded in preparations from 20-week-old male mice.

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Figure 4—CMs are impaired in diabetic Ins2+/Akita mice. The rate of action potential discharge evoked by 15-s force steps (A) and the meantemporal discharge pattern produced by 20-g steps (B) in Ins2+/Akita andWTCM fibers are shown.C: Application of force ramp stimuli evokeda significantly reduced rate of impulse discharge in Ins2+/Akita compared with WT CM fibers at 20 g, with a pattern of reduced response ratesat lower ramp forces (P = 0.035, Mann-Whitney U test). D: The mean firing patterns evoked by stimulation of Ins2+/Akita and WT CM fiberswith 20-g ramps show that diabetic CM fibers respond with reduced discharge rates throughout the stimulus, particularly in response tothe peak force at the end of the challenge. E: Typical traces of the response of Ins2+/Akita andWTCM fibers illustrate that diabetic CM fibersproduce fewer action potentials in response to the application of 20-g force ramps. *P, 0.05; compared with WT, Mann-Whitney U test.All fibers were recorded in preparations from 20-week-old male mice.

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the paw-pressure test. The distinct behavioral profilesobserved using different test modalities underscoresthe value of using a comprehensive behavioral testingstrategy.

MechanosensationPatients with diabetic neuropathy commonly reportnumbness, spontaneous pain, and dysesthesias (34), butloss of thermal (cold and heat detection) and mechanical

Figure 5—Temperature-evoked responses in mechanosensitive C-nociceptors. The distribution of temperature thresholds and thetemperature change required to elicit cold (A and B) and heat activation (F and G) in Ins21/Akita and WT CMs are shown. The number ofaction potentials evoked by a 60-s cold rampwas significantly reduced in CM nociceptors in preparations from Ins21/Akita compared toWTmice(C). The temporal response pattern evoked by cold ramps did not differ between fibers from Ins21/Akita and WT mice (D). Heat-evoked actionpotentials in CM nociceptors in preparations from Ins21/Akita and WT mice (H and I). Example traces of WT and diabetic CM nociceptorsresponding to cold (E) and heat (J). *P, 0.05, compared toWT, Mann-Whitney U test. The numbers of fibers studied in the different categoriesare indicated in panels D and I. All fibers were recorded in preparations from 20-week-old male mice.

1658 Sensory Neuropathy in the Ins2+/Akita Mouse Diabetes Volume 67, August 2018

(mechanical and vibration detection) sensation are thedominant sensory symptoms in patients with mild, severe,or no pain (3,35). Hypersensitivity to thermal or mechan-ical stimulation (evoked pain) can thus not be viewed astranslational measures of spontaneous pain or painfuldiabetic neuropathy. Importantly, we observed only a re-duced responsiveness of mechanosensitive and thermo-sensitive fibers here, in good agreement with an earlierskin-nerve investigation of mice made diabetic by strepto-zotocin (STZ) treatment (36).

Our electrophysiological studies demonstrate thatIns2+/Akita AM and, to a lesser extent, CM nociceptors

failed to sustain a high action potential discharge rate inresponse to intense mechanical stimulation. The forceactivation threshold of Ins2+/Akita AM fibers, but not CMfibers, was increased somewhat compared with fibers fromWT mice, whereas C fibers exhibited a degree of conduc-tion slowing. Together, these results suggest that neuronalexcitability and conduction, rather than transduction ofmechanical and thermal stimuli, are primarily affected bydiabetes in Ins2+/Akita mice. Our results are consistent withthe loss of mechanical nociception observed in mice in vivoand clinically in patients (1,37,38).

Earlier investigations of STZ-induced diabetic neu-ropathy in mice (36) demonstrated a reduced mechanicalsensitivity of RA fibers and a more pronounced loss of CMnociceptor activity than observed here in Ins2+/Akitamice. Itis possible that the longer interval between consecutivemechanical challenges used here may have allowed a fullerrecovery of CM fiber activity. Other possible reasons fordifferences between the two studies include the age ofdiabetes onset (which is very early in Ins2+/Akita), the dura-tion of diabetes, and the possibility that STZ affects cellsoutside the pancreatic islets (39–42).

Thermal NociceptionDiabetic Ins2+/Akita mice displayed an impaired sensitivityto noxious heat and cold in vivo and in vitro. Studies oftemperature-sensitive CM fibers (CMHs and CMCs) inisolated skin-nerve preparations from Ins2+/Akitamice dem-onstrated a markedly reduced responsiveness to cold, butnot to heat. DRG neurons from Ins2+/Akita mice showeda markedly reduced heat responsiveness compared withneurons from WT mice, but an unchanged cold sensitivity.The capsaicin receptor TRPV1 is the principal heat trans-ducer in DRG neurons, although other ion channels havebeen proposed to contribute to the transduction of nox-ious heat (e.g., during inflammation or in response to veryhigh temperatures) (43–46). A high concentration of theTRPV1 agonist capsaicin evoked [Ca2+]i responses in equalproportions inWT and Ins2+/AkitaDRG neurons, suggestingthat an altered expression of TRPV1 is unlikely to explainthe reduced heat sensitivity in Ins2+/Akita neurons. It ispossible that a reduced activity produced by, for example,glycation (47) or altered phosphorylation of TRPV1 maycontribute to the reduced sensitivity (48). It is likely thatheat-evoked [Ca2+]i responses are mediated in partby voltage-gated Ca2+ channels, after an initial TRPV1-mediated depolarization. An altered activity of voltage-gated channels or differences in membrane potential thatinfluence calcium entry via voltage-gated calcium channelsmay thus be responsible for the reduced heat sensitivityobserved in DRG neurons isolated from Ins2+/Akita mice.The reduced heat responsiveness of isolated DRG neu-rons, but largely unchanged activity of CMH fibers, mayindicate that mechanoinsensitive heat nociceptors areaffected by diabetes.

In contrast to the reduced heat responsiveness ofIns2+/Akita DRG neurons, we did not detect any deficits in

Figure 6—Temperature-evoked responses in DRG neurons. A: DRGneurons loaded with Fura-2 were challenged with cold and heatfollowed by the TRPM8 (transient receptor potential cation channelsubfamily M member 8) agonist icilin (1 mmol/L), the TRPV1 agonistcapsaicin (1 mmol/L), and KCl (50 mmol/L). The black trace is anexample of a typical icilin- and cold-sensitive neuron. This neuronexhibits an off-response after the heat ramp, when the temperatureis returned to ;30°C. The red trace is an example of a heat- andcapsaicin-sensitive neuron. B: Neurons that responded to a temper-ature ramp with a discontinuous rate of [Ca2+]i increase were con-sidered heat/cold sensitive and included in the analysis. Thedistribution of temperature thresholds for heat activation (C) andcold activation (E) in Ins2+/Akita and WT neurons. D: Heat-evoked[Ca2+]i response amplitudes were reduced in neurons from Ins2+/Akita

mice compared with WT mice, whereas the amplitudes of coldresponses were unchanged (F ). *P , 0.05, ***P , 0.001, unpairedt test. DRG neurons were isolated from male mice 12 or 20 weeks ofage.

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the neuronal response to cold ramps. We used a modestcooling rate in our experiments, and different rates of coolingare known to stimulate different sensory neuron populations(49,50). It is therefore possible that a subpopulation ofcold-sensitive Ins2+/Akita DRG neurons with altered prop-erties may have been overlooked.

Nociceptive AM and CM fibers, as well as mechanicallyinsensitive thermoreceptors terminate as free nerve end-ings in the epidermal layer of the skin (51,52). A reduceddensity of IENFs may thus have contributed to the im-paired thermal andmechanical nociceptive function in vivo.

Islet TransplantationTransplantation of pancreatic islets isolated from healthyWT mice, progressively improved blood glucose levelsand achieved normoglycemia after ;3 weeks. In contrast,the behavioral sensitivity to noxious mechanical and coldstimulation was restored to the WT level by the time offirst behavioral test (2–3 days after transplantation). Thisrapid recovery of sensory function may indicate thathyperglycemia is not directly responsible for maintainingthe impaired nociception. One possible explanation for therapid restoration of nociceptive function is that insulinitself may modulate sensory neuron function and act asa neurotrophic factor (reviewed in the study by Grote andWright [53]). A rapid neurotrophic or sensitizing effect ofinsulin on sensory neurons after transplantation is alsoconsistent with the observation that the insulin signal

transduction machinery is intact or even upregulated inZucker diabetic fatty rats with well-established neuropathy(54). Furthermore, local intraplantar administration ofinsulin in diabetic mice rapidly (within days) increasedthe density of IENFs, a time course that is consistent withthat observed here for the recovery of behavioral sensi-tivity in vivo after islet transplantation (55). In our experi-ments on isolated DRG neurons, we did not match theinsulin concentrations to the levels that the DRG neuronswould experience in vivo. The observed differences be-tween WT and Akita neurons can thus not be explained byany differences in the insulin concentrations in the cellculture medium. Importantly, this also means that thecompromised heat sensitivity of Ins2+/Akita neurons, whichis in line with our behavioral observations in vivo andelectrophysiological observations in vitro, are due to di-abetes, not simply due to differences in insulin signalingafter isolation of the neurons. It is likely that sensoryneurons exhibit heterogenous insulin signaling mecha-nisms, since the expression patterns of the insulin recep-tors IRS1 and IRS2 are not uniform across DRG neurons(56). The concentration of insulin present in the mediumhere (;4 pmol/L) is below the concentrations required toexert trophic effects on isolated sensory neurons (23).

Conclusions

Diabetes caused by Ins2C96Y is associated with impairednociception, loss of IENFs, a reduced sensory neuronal

Figure 7—Recovery from sensory neuropathy after islet transplantation. A: IENFs were visualized by immunostaining for PGP9.5. Scale bar,20 mm. B: The IENF density was significantly reduced in skin sections from male 20-week-old Ins2+/Akita mice compared with age-matchedWT littermates. Male diabetic Ins2+/Akita mice were transplanted at an age of 14–20 weeks. After islet transplantation (5–6 weeks), the IENFdensity recovered to the level seen in healthyWTmice.C: Blood glucose concentration was progressively restored after islet transplantation,reaching normoglycemic levels after 2–3 weeks. The sensitivity to noxious mechanical (paw-pressure test, D) and cold (cold-plate assay,E) stimulation rapidly increased to WT level after islet transplantation (compare with WT in Fig. 1C and E). Individual data points from each ofn = 5 transplantedmice are shown inC–E. The areas shaded in gray (C–E ) indicate the level in maleWT littermates (mean6 SD). Data in B arethe individual raw values, and the box indicates the median with interquartile range of n = 5–7 mice. *P , 0.05, **P , 0.01, ***P , 0.001,ANOVA followed by Tukey (compared with Ins2+/Akita; B) or Dunnett (compared with pretransplantation; C–E) post hoc test.

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heat sensitivity, and a reduced action potential dischargerate in mechanosensitive nociceptive afferent fibers in vitro.Functional and structural improvement from establisheddiabetic neuropathy was previously reported frommice thatrecovered spontaneously from STZ-evoked diabetes (57).Our results highlight the importance of distinguishingsensory abnormalities from the spontaneous pain thatmay be caused by diabetic neuropathy in patients. Islettransplantation restored IENF density (assessed at theend of the experiment, 5–6 weeks after transplantation).Remarkably, islet transplantation restored nociceptive func-tion before normoglycemia was established, indicatingthat lack of insulin, rather than hyperglycemia per se, isresponsible for the impaired nociception in diabetic mice.

Ins2+/Akita, and other strains carrying Ins2 mutations(58), are convenient and attractive models of type 1 diabetes.Here we show that diabetic neuropathy in the Ins2+/Akita

mouse is characterized by a loss of evoked sensoryresponses, consistent with the sensory profile most com-monly observed in patients with painful or painless neu-ropathy (3). Studies of experimental models that rely ondisease-relevant mechanisms and faithfully reproduce theclinical condition, such as the Ins2+/Akita mouse, are likelyto generate conclusions of translational predictive validity.Ins2+/Akita appears to have an advantage over several othercommonly used rodent models of diabetic sensory neurop-athy that are associated with mechanical hypersensitivity.The hypersensitivity seen in the latter models contrasts withclinical experience in humans where reduced tactile sensi-tivity is used to detect the onset of diabetic neuropathy.Although patients with painful diabetic neuropathy maydisplay a more marked loss of sensation than those withpainless neuropathy, sensory loss is the primary symptom ofboth groups (3,35,38). Sensory testing of diabetic rodents isthus appropriate as a sensitive experimental measure ofsensory neuropathy, but not as a translational measure ofspontaneous pain and dysesthesias.

Funding. This study was supported by the Diabetes UK Alec and Beryl WarrenAward to A.J.K., S.B., and D.A.A. (BDA 13/0004649). F.G. was supported by theJohannes und Frieda Marohn-Stiftung.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. N.V., F.G., C.G., A.L.A., and A.J.K. performed andanalyzed experiments and contributed to experimental design and manuscriptpreparation. S.B. and D.A.A. contributed to experimental design and manuscriptpreparation. D.A.A. is the guarantor of this work and, as such, had full access to allthe data in the study and takes responsibility for the integrity of the data and theaccuracy of the data analysis.

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