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Figure  1  Representa5ve  epifluorescence  graphs  of  cultured  rat  DRG  neurons  before  (A)  and  aCer  (B)  applica5on  of  100nM  capsaicin.  Arrows,  Fura-­‐2-­‐loaded  cells  exhibi5ng  Ca2+    mobiliza5on  in  response  to  capsaicin.  (Scale  bars,  30μm.)  

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Figure  2  Representa5ve  traces  of  capsaicin  evoked  calcium  responses  in  rat  DRG  neurons  (Blue),  enhanced  by  laminectomy  (Red).    Intrathecal  pretreatment  with  the  TRPV1  agonist  RTX  a^enuated  laminectomy  induced  enhancement  (Green).    

Figure  3    On  POD1,  potassium  chloride  evoked  calcium  transients  in  rat  DRG  neurons  as  measured  by  the  ra5o  difference  between  the  peak  ra5o  and  baseline  ra5o  (∆Ra5o  340/380).  Data  are  presented  as  mean±SEM  of    ∆Ra5o  340/380.  ***p<0.001,  **p<0.01,  n=50-­‐80  cells  from  4-­‐6  rats  each  group.  

Figure  4  On  POD1,  dura5on  (seconds)  of  recovery  to  80%  of  the  baseline  of  K+  evoked  Ca2+  transients  in  rats  DRG  neurons.  Data  are  presented  as  mean±SEM  of  the  5me  dura5on  to  80%  recovery  of  baseline  of  ∆Ra5o  340/380.  ***p<0.001,  **p<0.01,  n=  50-­‐80  cells  from  4-­‐6  rats  each  group.  

Figure  5  On  POD1,  capsaicin  evoked  calcium  transients  in  rat  DRG  neurons  as  measured  by  the  ∆Ra5o  340/380.  Data  are  presented  as  mean±SEM  of    ∆Ra5o  340/380.  ***p<0.001,  *p<0.05,  n=45-­‐70  cells  from  4-­‐6  rats  each  group.  

Figure  6  On  POD1,  dura5on  (seconds)  of  recovery  to  80%  of  the  baseline  of  capsaicin  evoked  Ca2+  transients  in  rats  DRG  neurons.  Data  are  presented  as  mean±SEM  of  the  5me  dura5on  to  80%  recovery  of  baseline  of  ∆Ra5o  340/380.  ***p<0.001,  *p<0.05,  n=  45-­‐70  cells  from  4-­‐6  rats  each  group.  

Figure  7  On  POD1,  percentage  of  capsaicin  responsive  DRG  neurons.  Data  are  presented  as  mean±SEM  of  the  percentage  of  neurons  responsive  to  capsaicin  in  the  whole  DRG.  ***p<0.001,  *p<0.05,  n=  45-­‐70  cells  from  4-­‐6  rats  each  group.  

Figure  8  On  POD7,  potassium  chloride  evoked  calcium  transients  in  rat  DRG  neurons  as  measured  by  the  ra5o  difference  between  the  peak  ra5o  and  baseline  ra5o  (∆Ra5o  340/380).  Data  are  presented  as  mean±SEM  of    ∆Ra5o  340/380.  **p<0.01,  n=45-­‐70  cells  from  4-­‐6  rats  each  group.    Figure  9  On  POD7,  dura5on  (seconds)  of  recovery  to  80%  of  the  baseline  of  K+  evoked  Ca2+  transients  in  rats  DRG  neurons.  Data  are  presented  as  mean±SEM  of  the  5me  dura5on  to  80%  recovery  of  baseline  of  ∆Ra5o  340/380.  **p<0.01,  n=  45-­‐70  cells  from  4-­‐6  rats  each  group.    

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Figure  10  On  POD7,  capsaicin  evoked  calcium  transients  in  rat  DRG  neurons  as  measured  by  the  ra5o  difference  between  the  peak  ra5o  and  baseline  ra5o  (∆Ra5o  340/380).  Data  are  presented  as  mean±SEM  of    ∆Ra5o  340/380.  ***p<0.001,  n=40-­‐60  cells  from  4-­‐6  rats  each  group.    Figure  11  On  POD7,  dura5on  (seconds)  of  recovery  to  80%  of  the  baseline  of  K+  evoked  Ca2+  transients  in  rats  DRG  neurons.  Data  are  presented  as  mean±SEM  of  the  5me  dura5on  to  80%  recovery  of  baseline  of  ∆Ra5o  340/380.  ***P<0.001,  **p<0.01,  n=  40-­‐60  cells  from  4-­‐6  rats  each  group.    Figure  12  On  POD7,  percentage  of  capsaicin  responsive  DRG  neurons.  Data  are  presented  as  mean±SEM  of  the  percentage  of  neurons  responsive  to  capsaicin  in  the  whole  DRG.  ***p<0.001,  n=  40-­‐60  cells  from  4-­‐6  rats  each  group.    

On  pod6  7  there  is  a  sig  diff  between  RTX  and  no  drug  laminectomy  in  running  distance  (two  way  anova,  for  mul5ple  t-­‐  test,  day  3,4,5,6,7  is  different.  P=0.016,  0.0013,0.0013,0.0002,0.0004).  On  pod0,  there  is  a  diff  between  RTX  and  no  drug  in  maximum  speed(two  way  anova,mul5ple  t  test,  day  0,1,2,  p=0.0003,0.01,0.02).  there  is  no  diff  between  the  2  group  in  accelera5on.  

there  is  a  sig  diff  between  RTX  and  no  drug  laminectomy  in  fine  motor  movement  (two  way  anova,  for  mul5ple  t-­‐  test,  day  1,2  is  different.  P=0.014,  0.026).  there  is  a  diff  between  RTX  and  no  drug  in  rearing(two  way  anova,mul5ple  t  test,  day  1,  p=0.008).  there  is  no  diff  between  the  2  group  in  accelera5on.  

Figure  13  On  baseline,  post  RTX  injec5on  day  7,  post  RTX  injec5on  day  14,  thermal  pain  withdrawal  threshold  with  a  cutoff  of  25  seconds.  *p<0.05,  n=4  rats  each  group.  

Prolonged and enhanced rodent intracellular Ca2+ signal in

dorsal root ganglion after laminectomy was shortened and down

regulated by intrathecal resiniferatoxin

Abstract

Background: postoperative pain is a challenging problem in clinic, compromising

quality of life and leading to extended hospital stay and economic burden and could

develop into chronic pain. Patients report significant pain and hypersensitivity

postoperatively. This study examined the capsaicin and high potassium induced

intracellular calcium response in cultured adult rat DRG neurons on postoperative day

1 and postoperative day 7, and examined the effects of resiniferatoxin treatment on

neuronal intracellular calcium responses and rats behavior including wheel running and

spontaneous home cage activities.

Results: Calcium imaging showed that laminectomy resulted in prolonged duration and

elevated amplitude of high potassium and capsaicin induced DRG neuron intracellular

calcium responses. Also the surgery led to higher responsive ratio in DRG populations,

compared to controls; this was reduced by intrathecal administration of 200ng

resiniferatoxin. Laminectomy resulted in decreased wheel running distance, maximum

speed , home cage rearing and fine movements. Resiniferatoxin partially reversed the

effects of laminectomy on wheel running and home cage spontaneous activities. It also

extended the heat response in hind paw after intrathecal injection.

Conclusions: Laminectomu induces TRP sensitization and higher response to high

potassium in DRG neurons and leads to reduced wheel running and spontaneous

activities in home cage. These effects may be mitigated by preoperative treatment with

TRPV1 agonist resiniferatoxin which could lead to desensitization of neurons, increase

wheel running and alleviate spontaneous pain.

Introduction

A large body of evidence point to the importtant role of TrpV1, the capsaicin receptor, in

sensation of noxious heat (Caterina et al., 1997). TrpV1is also believed to play a role in

increased

pain sensitivity following inflammation at least in part because inflammation induced

phosphorylation of TrpV1 results in increased sensitivity of this ion channel to

temperature (Bhave et al., 2002; Cesare et al., 1999; Nilius et al., 2005; Numazaki et

al., 2002). Attention has focused on TrpV1 and TrpV1 cells for their role in pain

sensation and as targets for pain therapies. Notably, TrpV1 antagonists have been

shown to be efficacious in animal models of chronic pain(Szallasi et al.,2007;Wong

andGavva,2009) but, as yet, have not been exploited in human pain management.

An alternate strategy for pain management involves ablation of TrpV1-expressing cells

or TrpV1 fibers. This approach has a long history: more than 25 years ago, capsaicin

injection was reported to dramatically decrease the level of inflammatory mediators

(e.g., substance P) and pain sensation for the life of animals (Jancso et al., 1977; Nagy

et al., 1981; Yaksh et al., 1979). More recently, the potent TrpV1 agonist, resiniferotoxin

(RTX) has been demonstrated to kill TrpV1-expressing neurons by affecting long-term

Ca2+ entry (Olah eal., 2001). RTX has also been used therapeutically in animals and

has been reported to alleviate chronic pain with no effects on discriminative touch or

proprioception (Brown et al., 2005; Karai et al., 2004; Neubert et al., 2003). In a rat

model, RTX was shown to eliminate many TrpV1 neurons, dramatically decrease

responses to capsaicin and thermal pain (Karai et al., 2004), and affect

mechanosensation (Tender et al., 2008).

Mishra et al 1 carried out a detailed investigation into the efficacy of RTX in ablating

sensory neurons and demonstrate that although a large subset of TrpV1neurons are

killed by a single injection, other TrpV1 cells are not affected even by repeated RTX

treatments. RTX reduces but does not eliminate capsaicin responses and thermal

sensation of normal mice.

We hypothesized that intrathecal application of RTX may block the DRB neurons

temporarily and reduce the pain behavior. RTX is supposed to work on trpv1 positive

small diameter neurons and leave intact the large diameter axons responsible for

proprioceptive and somatic sensations.The intrathecal approach is proposed to broadly

affect the DRGs surrounding the L4, L5 and their corresponding dermatome including

the laminectomy site to mediate a selective antinociceptive effects. By modifying the

trpv1 positive DRG neurons, this treatment can help relieve different clinical pain

including the postoperative pain.

Here, we set out to determine the function of TrpV1 cells and explored the use of RTX

as a means to affect the intracellular calcium response to capsaicin and hight potassium

stimulations. And to see if it can selectively eliminate trpv1 positive neurons in rats. The

primary aim of this study was to determine whether incisional pain produced a higher

calcium response in DRG neurons and how long it will last. The other aim of the study

was to determine if intrathecal RTX will promote the reduction of calcium response in

these DRG neurons after laminectomy and to check the effect of RTX on the hind paw

thermal response and the effect of RTX on wheel running and rodent spontaneous pain

behavior after intrathecal application. Low doses of RTX (100-250ng) has been proved

to be effective when given perineurally2, in single ganglionic injection or intrathecally3.

The minimus effective doses should be determined to avoid the po tential side effects

considering the clinical settings. So this study used the 200ng RTX to block multiple

ganglia by applying intrathecally.

Materials and Methods

Animals

Experiments were conducted in adult male Sprague-Dawley rats (Harlan,Indianapolis,

IN, USA) weighing 225-250 g. Animals were housed in groups of 2, maintained in a 12-

hour light-dark cycle with a temperature-controlled environment and given food and

water ad libitum. All procedures used in these experiments were reviewed and

approved by the Institutional Animal Care and Use Committee at the University of Iowa.

Animals were cared for and used in accordance with guidelines of the Committee for

Research and Ethical Issues of the IASP4.

Chemicals and reagents

1,1'-Dioleyl-3,3,3',3'-Tetramethylindocarbocyanine methanesulfonate (DiI), Fura-2

acetoxymethyl (AM) ester and Pluronic F-127 (stock concentration 20%) were

purchased from Invitrogen (Carlsbad, CA, USA). Concentrations were based on results

from previous studies5. DiI was dissolved in DMSO (25 mg/ml). Fura-2 AM (2.5µm) was

dissolved in 1mM dimethyl sulfoxide and stored at -20°C. When ready to use, the

Fura-2AM was diluted in bath solution (in mM: 130 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10

Hepes, 10 glucose, and pH adjusted to 7.4, and osmolality adjusted with sucrose to

310-325 mOsm), together with Pluronic F-127 (0.025%) immediately before use.

Resiniferatoxin (RTX), (Sigma Aldrich, St Louis, MO, USA) was dissolved in 10/10/80%

mix of ethanol, Tween-80, and saline. Capsaicin (Sigma, St. Louis, MO, USA) was

diluted to a final concentration of 1mM in 1.5% Tween-80, 1.5% alcohol and 97% saline.

Capsaicin (100nM) was diluted in normal bath solution immediately before use. All other

compounds were dissolved in normal bath solution directly. For obtaining high-

potassium extracellular solution (K+, 30mM), the K+ in normal bath solution was adjusted

to 30 mM by iso-osmotic replacement of Na+.

Labeling DRG neurons

Prior to tissue harvest (>5 days), sensory neurons innervating the lower back region

were labelled with the retrograde tracer DiI. General anesthesia was induced with

isoflurane (Abbott Laboratories, North Chicago, IL, USA) in room air and maintained at

1.5%, DiI solution (8 µl) was injected onto the right L4 and L5 laminae and surrounding

muscles (4 µl ×2) by positioning the needle 2mm away from the midline (the L4 and L5

spinous processes) with a 30 g injection needle.    

Intrathecal injection of RTX or Saline

Under 1.5% isoflurane anesthesia, each rat in the RTX group received intrathecal

(between the L4 and L5 vertebrae) injection of 20 µl RTX (200ng in 20 µl saline) using a

Hamilton syringe and a 25-gauge needle. Each rat in saline group received injection of

20 µl saline.

Behavioral pain tests

Rats in the RTX group were tested on baseline, post injection day 7 and post injection

day 14. For withdrawal latency to radiant heat stimuli, unrestrained rats were individually

placed onto a 3-mm thick glass surface (40 × 40 cm) under a plastic chamber (21 ×

27×15 cm) for approximately 15 minutes of acclimation. A focused radiant heat source

was applied from underneath the glass floor onto the center of hind paw. The latency to

evoke withdrawal was determined with a cut-off value of 30 seconds to prevent radiant

heat evoked injury to the hindpaw. Each rat was tested 3 times, with a 5-minute interval

between tests. The mean paw withdrawal latency was determined from 3 tests.

Guarding pain was performed before withdrawal tests to avoid the potential influence of

withdrawal responses on guarding pain.

Wheel running and home cage locomotion tests

Rats were placed individually in cages fitted with exercise wheels (Intellibio Activity

Wheel, Seichamps, France). The rats were housed individually and allow to run on the

exercise wheels with a computer that recorded the distance, speed, acceleration,

revolutions and time (in one hour bins). A stable baseline was achieved after 14 days of

running wheel exposure. The last 3 days were averaged to obtain the baseline. Data

was recorded daily for 14 days. The distance traveled by the rat, acceleration, maximum

speed, total motor movement, and rearing activity of rats became stable after 2 weeks

habituation with this experimental protocol. The effects of a treatment on each rat were

analyzed as percentage of the distance traveled during the baseline.

Home Cage Activity Activity during the dark cycle (period when rats are active) after

surgery was monitored. After acclimatization to individual home cages (17 x 5 x 18 inch)

for 3 days, the cages were inserted into a SmartFrame Cage Rack Motor Monitor

System (Kinder scientific, Poway, CA). Two grids of 7x15 infrared beams surrounded

the cage; one was placed 6 cm above the floor to record total locomotor activity as total

beam interruptions. The second series of beams was placed 18 cm above the cage

floor and count rearing, when the rat stood on its hindpaws. Beam breaks were counted

to record total activity and rearing. The data was then transmitted to a host computer

and collected in 1-hour bins. The last 3 days were averaged as the baseline, and then

rats would undergo sham surgery or full laminectomy. We used these monitors to

examine hypermobility by acute pain stimuli.

Laminectomy Procedure

After the induction of general anesthesia with isoflurane in room air and maintained with

1.5% isoflurane. The surgery was performed with spontaneous ventilation through a

nose cone. Each animal was shaved and sterilized with povidone iodine at the incision

site. A 4cm midline longitudinal incision is made with a # 11 blade through the skin

down to the fascia between L3-L6. Blunt dissection was continued through the incision

into the muscle (removing muscle away from bone) to further divide and retracts the

muscle. The posterior bony spine was exposed and L4-L6 spinous processes removed.

The bone between L4-L5 was removed using a battery-powered drill. The dura was

exposed without breaching it. After surgery, the wound was closed in layers; the muscle

was closed with 4 sutures, followed by fascia using absorbable 3-0 vicryl suture

(Ethicon, San Angelo, TX, USA). The skin was closed with 4 sutures of 3-0 nylon

(Ethicon, San Angelo, TX, USA).

Cell Culture

Rats were euthanized with 100% carbon dioxide and the right-sided L1-L3 DRG were

surgically removed and desheathed in ice-cold Hanks balanced salt solution (HBSS)

(Gibco, Gaithersburg, MD, USA). DRGs were incubated 20 min at 37°C in 3 ml HBSS

containing 0.125% papain (Worthington, Lakewood, NJ, USA) and then centrifuged at

800 g for 1 min. The papain solution was removed and replaced and incubated for 20

min with 3ml HBSS containing 10 mg collagenase IV (Worthington, Lakewood, NJ,

USA) and 14mg dispase (Invitrogen, Carlsbad, CA, USA). DRGs were then

centrifuged at ∼800 g for 1 min, resuspended with culture media F12 (Gibco,

Gaithersburg, MD, USA). DRGs were mechanically dissociated in this solution with fire-

polished Pasteur pipettes and then plated onto glass coverslips, previously coated by a

solution of 0.1 mg/ml poly-D-lysine (Sigma Aldrich, St Louis, MO,USA) and 5 µg/ml

mouse laminin (BD Bioscience, Bedford, CA, USA). A 0.5-mm-thick silicone ring with a

4-mm-diameter opening sealed to the surface of each well reduced the plating area in

each well. The cells were incubated at 37°C, in 5% CO2 for 2 h. After 2 h, the wells were

filled with 2ml F12 media (Gibco, Gaithersburg, MD, USA) containing 10%(v/v) Fetal

Bovine Serum (FBS), 50 U/ml penicillin , 50 µg/ml streptomycin, and 50ng/ml nerve

growth factor (NGF) (Sigma Aldrich, St Louis, MO,USA), and incubated overnight.

Subclassification of DRG neurons

Neurons were divided into small (<30 µm), and large (≥30 µm) based on cell body

diameter as measure with a calibrated eyepiece reticule. Only neurons with visible

nucleus are used. Neurons with a small cell body diameter are likely to be nociceptive

afferents, while those with a large cell body diameter are likely to be non-nociceptive

afferents6.

Intracellular Ca2+ measurements

All experiments were performed within 24h of tissue harvest. DRG neurons were loaded

with the calcium indicator dye, Fura-2 AM ester (2.5µM) along with 0.025% Pluronic for

20 min at room temperature as previously described 5. Following fura-2 loading, DRG

neurons were placed in a recording chamber mounted on the stage of an inverted IX-71

microscope (Olympus IX71, Japan) and were continuously perfused for 5 minutes with

normal bath solution before the experiment began. Fluorescence data were acquired on

computer running Slidebook 5.0 software (Molecular Devices, Sunnyvale, CA, USA) at

20°C. Only cells with DiI label were used for calcium imaging. Fluorescence was

alternately excited at 340nm and 380nm using the Lambda DG-4 filter changer (Sutter

Instruments, Novato, CA, USA) via a 10x (Numerical Aperture NA=0.30) or 20x

(NA=0.70) objectives. Emitted fluorescence was collected at 510nm using an ORCA-

03G cooled digital CCD camera (Hamamatsu Photonics, Japan). Pairs of images were

sampled at 1-3 Hz. For each field, an average of 20-40 cells for the 20x objective were

tracked in real time. The ratio of fluorescence emission (510 nm) in response to 340/380

nm excitation was acquired at 1-3 Hz during drug applications. All drugs were applied

via a gravity-fed chamber perfusion system. Cells were depolarized by capsaicin (100

nM, 30 seconds) or K+ (30 mM, 30 seconds). Cells not responsive to K+ were excluded.

Two properties of calcium influx were measured: peak ratio (340/380 nm) of the Fura-2

signal, and duration of the Fura-2 signal (calculated as time to 80% of baseline value).

In case of capsaicin depolarization, the percentages of cells responsive to capsaicin

were also compared between groups.

Statistical analysis

Data are expressed as mean± standard error of the mean (SEM) unless otherwise

stated. The “n” for each group refers to the number of neurons. Statistical significance

was determined using Student’s t-test, or ANOVA when appropriate followed by Tukey’s

post-hoc comparisons (GraphPad Prism software, San Diego, CA, USA). Differences at

the P<0.05 level were considered statistically significant.

Results

To assess the effect of laminectomy on the DRG neurons’ intracellular calcium

transients’ responses to K+ or capsaicin induced depolarization, Fura-2 Ca2+ imaging

experiments were performed on DRG neurons that were transiently depolarized with

30nM KCl or 100nM capsaicin. We only analyzed the L1-L3 DRG DiI+ neurons which

were supposed to represent the neurons innervating the laminectomy area at the lower

back region 7. And only the calcium transients of the potassium responsive neurons

were analyzed.

On POD1, laminectomy potentiates K+ evoked Ca2+ transients in DRG neurons

and RTX attenuated the Ca2+ transients enhancement induced by laminectomy.

The magnitudes of K+ evoked Ca2+ transient were analyzed using the change of peak

ratio to baseline. And the decay was measured as time from onset to return to 20% of

peak (T80). Results of this analysis for DiI+ neurons revealed significant effects

associated with laminectomy (P<0.001). When using K+ to depolarize the DRG

neurons, both the magnitude and the decay of Ca2+ transients in DRG neurons were

enhanced by laminectomy. Laminectomy induced higher amplitude (laminectomy

1.70±0.07vs. sham 1.26±0.09, ***p<0.001, Fig.1) and longer decay (laminectomy

269.21±34.98 vs. sham 137.6±15.55, ***p<0.001, Fig.2) when compared with sham

group. Laminectomy significantly enhanced the Ca2+ transients evoked by 30nM KCl

depolarization and attenuated the recovery from depolarization evoked by 30nM KCl.To

determine whether RTX can have effect on DRG neurons’ K+ evoked Ca2+ transients,

we administered 200ng RTX intrathecally at L4 and L5 level 7 days before

laminectomy.Post-hoc (Tukey)analysis indicated that intrathecal 200ng RTX

pretreatment reduced the K+ evoked calcium flux in laminectomy group. (laminectomy

1.70±0.07, vs. RTX and laminectomy 1.14±0.07, *** p<0.001, n= 46 vs 31 cells, 6 rats,

Fig. 1), while the decay of the K+ evoked Ca2+ transient was shortened by RTX

pretreatment (laminectomy 269.2±34.98, vs. RTX and laminectomy 82.43±11.12, ***

p<0.001, n= 25 vs 21cells, 6 rats, ***P<0.001, Fig. 2). Thus, potassium-depolarization

mediated calcium mobilization in laminectomy group was drastically reduced in the

presence of RTX. On POD1, RTX also can reduce the amplitude of calcium flux in sham

rat DRG neurons. (sham 1.26±0.09, vs. RTX and sham 0.88±0.07, **p<0.01, n= 45 vs

44cells, 6 rats, Fig.1). and shortened the duration. (sham 137.7±15.55, vs. RTX and

sham 74.46±8.29, *p<0.05, n= 45,37 cells, 6 rats, Fig.2). Therefore, on POD1, in both

laminectomy rats and sham rats, RTX reduced the calcium response to KCl , either by

reducing the amplitude of calcium peak or by shortening the decay of the transient.

On POD1, capsaicin evoked Ca2+ transients in DRG neurons was also potentiated

by laminectomy, which was attenuated by intrathecal RTX.

Laminectomy induced higher amplitude when compared with sham group (laminectomy

2.60±0.14 vs. sham 2.03±0.14, *p<0.05, Fig. 3 Post hoc analysis (Tukey test)) and slow

the recovery of transients in small diameter neurons (laminectomy 986.10±116.10, vs.

sham 511.31±78.65, ***P<0.001, Fig. 4). And there is also a significant increase in the

percentage of capsaicin responsive neurons in the DRG populations induced by

laminectomy (laminectomy 75.20±3.79% vs. sham 63.67±1.93%, *P<0.05, Fig. 5).

As observed RTX effect on potassium induced calcium transients, there was also a

statistically significant effect of pre-surgery RTX in both the magnitude (laminectomy

2.60±0.14, vs. RTX and laminectomy 1.52±0.16, *** p<0.001, n= 40 vs 34 cells, 6 rats,

Fig. 3)and decay (laminectomy 986.10±116.10, vs. RTX and laminectomy

378.70±69.18, *** p<0.001, n= 32 vs 27 cells, 6 rats, Fig. 4) of the capsaicin evoked

Ca2+ transient in small DiI+ DRG neurons. In addition, RTX decreased the percentage

of capsaicin responsive DRG (laminectomy 75.20±3.79% vs. RTX 51.00±3.67%,

***p<0.001, Fig. 5). It also decreased the percentage of capsaicin responsive DRG in

sham rats (sham 63.67±1.93% vs. RTX and sham 33.10±1.78%, ****P<0.0001).

On POD1, in both laminectomy rats and sham rats, RTX reduced the calcium response

to capsaicin, either by completely eliminating the transient or by reducing the amplitude

of calcium peak or by shortening the decay of the transient.

On POD7, laminectomy did not produce significant changes in K+ evoked Ca2+

transients in DRG neurons either in amplitude (laminectomy 1.04±0.11vs. sham

1.18±0.08, p>0.05,n= 23 vs 40 cells, 4 rats Fig. 6) or decay(laminectomy 108.70±26.31

vs. sham 116.2±12.79, p>0.05, Fig. 7).

On POD7, laminectomy extended the decay of capsaicin evoked Ca2+ transients.

And RTX attenuated the laminectomy enhanced capsaicin response in DRG

neurons

On POD7, even the magnitude of capsaicin evoked Ca2+ transients did not differ

between laminectomy group and sham group(laminectomy 2.19±0.18 vs. sham

1.77±0.15,p>0.05, n=20 vs 38 cells, 4 rats each group Fig. 8) although RTX decrease

the amplitude both in laminectomy rats (laminectomy 2.19±0.18, vs. RTX and

laminectomy 1.21±0.12, n=20 vs 37 cells , 4 rats each group,*** p<0.001, Fig. 8) and

sham rats(sham 1.77±0.15% vs RTX and sham 0.85±0.09%, n=38 vs 30 cells, 4 rats

each group, ***P<0.001, Fig. 8).

However there was still increased decay of capsaicin evoked Ca2+ transients in

laminectomy group (laminectomy 1044±149 vs sham 414±44.76 seconds, n= 25vs 33

cells, 4 rats each group, ***P<0.001, Fig. 9), and RTX shortened the decay in both

laminectomy rat (laminectomy 1044.0±149.3 vs RTX and laminectomy 188.6±33.68

seconds, n=25 vs 26 cells, 4 rats each group, ****P<0.0001, Fig. 9) and sham

rats(sham 414±44.76% vs RTX and sham 161.2±11.89%, n=33 vs 35 cells, 4 rats each

group, *P<0.05, Fig. 9).

On POD7, there was no significant difference in terms of the percentage of capsaicin

responsive neurons between the sham group and laminectomy group (laminectomy

71.75±6.96% vs. sham 59.51±2.82%, 4 rats each group, p>0.05, Fig. 10), even when

RTX still lowered the percentage of capsaicin responsive DRG neurons both in RTX

pretreated laminectomy group (laminectomy 71.75±6.69% vs RTX laminectomy

45.0±2.04%, 4 rats each group,**p<0.01, Fig. 10) and sham rats(sham 59.51±2.82% vs

RTX and sham 33.60±1.50%, n=10 vs 5 rats each group, *P<0.001, Fig. 10).

Thus, on POD7, RTX still attenuated the laminectomy enhanced DRG neurons’

responses to capsaicin.

Effects of RTX on wheel running and home cage activity

We observed the whole week after laminectomy using RTX. And found significant

changes after RTX administration. The attenuated behavior was recovered faster

compared with laminectomy alone. Including running distance and locomotion.

r tx la m i D is ta n c e

B as e line 0 1 2 3 4 5 6 7

0

5 0

1 0 0

1 5 0

2 0 0R T x + L a m in e c to m y lia n g

N o D ru g (N o A lz e t) L a m i lia n g

S h a m lia n g

r tx la m i M a x S p e e d

B as e line 0 1 2 3 4 5 6 7

0

5 0

1 0 0

1 5 0R T x + L a m in e c to m y

L a m i

S h am

wo    

On  pod6  7  there  is  a  sig  diff  between  RTX  and  no  drug  laminectomy  in  running  distance  (two  way  anova,  

for  multiple  t-­‐  test,  day  3,4,5,6,7  is  different.  P=0.016,  0.0013,0.0013,0.0002,0.0004).  On  pod0,  there  is  a  

diff  between  RTX  and  no  drug  in  maximum  speed(two  way  anova,multiple  t  test,  day  0,1,2,  

p=0.0003,0.01,0.02).  there  is  no  diff  between  the  2  group  in  acceleration.  

R T X F in e m o to r m o v e m e n t

B a s e line 0 1 2 3 4 5 6 7

0

5 0

1 0 0

1 5 0S h am

L a m in e c to m y

L a m in e c to m y -R T x

R T X R e a r in g

B as e line 0 1 2 3 4 5 6 7

0

5 0

1 0 0

1 5 0S h a m lia n g

L a m in e c to m y

L a m in e c to m y R T x

there is a sig diff between RTX and no drug laminectomy in fine motor movement (two

way anova, for multiple t- test, day 1,2 is different. P=0.014, 0.026). there is a diff

between RTX and no drug in rearing(two way anova,multiple t test, day 1, p=0.008).

there is no diff between the 2 group in acceleration.

Effects of RTX on the thermal hyperalgesia

The effects of the intrathecal administration of RTX (200ng) on thermal hyperalgesia in

naïve rats at 7 days and 10 days after injection of RTX were investigated.

Our results show that the intrathecal administration of 200ng RTX inhibited the thermal

response of hindpaw (Fig. 11), which confirmed the valid intrathecal injection of RTX.

Discussion

The pain after operation represents a significant clinical challenge for treatment. The

search for new treatment options of postoperative pain has been directed toward

specific mediators and modulators of pain and inflammation.

In this study, we showed that laminectomy sensitized the DRG sensory neurons

indicated by elevated amplitude of calcium response curve and prolonged duration both

in response to high K and capsaicin and a higher percentage of capsaicin responsive

,TRPV1 positive neurons.

The present study also showed that it RTX attenuated DRG responsiveness to

potassium and capsaicin evidenced by recovered amplitude, reversion of prolonged

duration to high K and capsaicin and reduced percentage of TRPv1 positive DRG

neurons.

Furthermore, on pod7 laminectomy still has prolonged the duration of intracellular

neuronal calcium response to capsaicin, which is also reversed by pretreatment of i.t.

RTX indicated by a normalized duration of calcium response. This showed an extended

effect of RTX.

Laminectomy produces a decrease in rat wheel running and spontaneous home cage

activities. I .T. RTX pretreatment reversed the decrease of wheel running distance and

recovered the rearing and fine movement. Because there were no sided effects

observed in the experiment, thus the safety profile of RTX was established. We propose

that RTX intrathecal application could be a useful strategy in alleviating postoperative

pain.

We developed an rat model of postoperative pain and examined whether TRPV1 play a

role in mediating postoperative pain injury. In sensory dorsal root ganglia, the trpv1 are

expressed on small and medium –sized neurons and are mainly involved with mediation

of nociceptive information. TRPV1 is expressed by the type II A-delta and C-fiber

neurons, functioning as a molecular integrator of nociception. TRPV1-positive neurons

are essential for the development of mechanical allodynia.

Trvp1 is sensitized by phosphorylation of trpv1 via cAMP8. and we tested he effect of

specific TRPV1 agonists could overexcited DRG neurons and provide a potential way of

relieving pain.

Prior studies have shown that pain could induce expression of TRPV1 and neuron

phenotype change. Nerve injury and inflammation could enhance the expression of

trpv1 including the transcription, translation, post translational modification and

trafficking of trpv1(Hucho T 2007 neuron, zhang x, EMBO J 2005, Ji RR, Neuron 2002).

The net effect is central and peripheral sensitization. This could switch the phenotype of

DRG nruosns and spinal cord neurons from naïve state to an altered state. Thus

redcuce the pain threshold(Patapoutian A, 2009).Trpv1 could enhanced form gene

level, translational level (mRNA), and post-translational modifications such as

phosphorylation, n-glycosylation and covalent modification9. Especially phosphorylation

by PKCᵋ (Cesare 1999, Mandadi S 2006 pain) 10and PKA(Bhave 2002, Lopshire, 1998).

Also there is evidence showing the phosphorylation of Trpv1 is enhanced. .Inflammaorty

mediators sensitized trpv1 through phosphorylation of trpv1 by protein kinase PKCᵋ

(Mandadi S 2006 Pain zhang H, 2007 J neuroscience).

In postoperative rat, trpv1 could be elevated because of gene level, rna level and

posttranstlational level change. Recent study demonstrated that TRpv1 antagonist

intratheally and local application both can allevievate thermal hyperalgsiea and

mechanical allodynia(Uchytilova mole pain ,2014 pure behavior). Also in a model of

thoracotomy postoperative pain, RTX significantly reduced mechanical allodynia

following surgery.(Shin Jw , 2010). TRPV1-expressing C-fibers are sensitized to heat

and acid after incision (Kang S 2010 Pain), All this indicated the involvement of trpv 1 in

postoperative pain. As we have demonstrated here, there were marked differences in

small DRG neurons between laminectomy group and sham group with respect to the

magnitude and decay of capsaicin-evoked Calcium transients. In order to identify

neurons innervating the site of surgery, DiI was injected in lumbar region prior to the

surgery. One day after surgery, there was a significant increase in the magnitude and

decrease in the decay of capsaicin evoked calcium transients in small DRG neurons.

Similar decreases in decay were also observed in the transients evoked by capsaicin

even 7 days after laminectomy. The reason of the enhanced intracellular calcium

transients during inflammation could be the inflammatory mediators such as nerve

growth factor because they might affect the calcium efflux, uptake and binding of

calcium and thus contribute to the enhanced calcium transients during inflammation (Lu,

2008 ). The trpv1 sensitaztion in laminectomy group could be attributed to inflammatory

mediators such as Nerve growth factor (NGF) because it induces sensitization of the

TRPV1(Zhu W, 2007 mol cell neurosci). Other Inflammatory mediators (BK, substance

P) also modulate TRPV1 activity (Sugiura T. Bradykinin lowers the threshold

temperature for heat activation of vanilloid receptor

1. J Neurophysiol 2002; 88:544–8. Neurokinin-1 receptor enhances TRPV1 activity in

primary sensory neurons via PKCepsilon: a novel pathway for heat hyperalgesia. J

Neurosci. 2007 Zhang H). Studies have shown that trpv1 sensitization(by heat)

produced a larger membrane deploarizations in rat DRGs. This could lower the

threshold and increase excitability of DRG neurons. (Neelands, 2008 inflam research,

zhang xf, 2011). We demonstrated that surgery induced an enhanced calcium response

to both potassium and capsaicin. Because Intracellular calcium plays a critical role in

mediation of many cellular processes such as transcription, ion channel activity, it

regulates the neuronal excitability and transmitter release (Berridge, 2005)and hormone

release, and Ca2 +-dependent gene transcription. , so the enhanced calcium response

seen in small DRG after surgery might contribute to the postoperative pain by changing

afferent excitability and facilitating transmitter transcription and release from the

nociceptive afferents. Indeed, pervious study showed that activation of trpv1 in afferent

terminals could increase the rate of glutamate release by allowing calcium into neurons

(Shoudai 2010). Such results suggest that activated, presynaptic TRPV1+ receptors

trigger continuous resting release of glutamate vesicles at physiological temperatures

only in capsaicin-responsive terminals. Thus the sensitization of DRGs found in

postoperative pain model indicated the higher excitability of DRG neurons and

increased neurotransmitter release in afferent terminals.

In terms of percentages of trpv1 response positive cells, unregulated trpv1

responsiveness in primary-afferent fibers occurs in inflammatory disease and irritable

bowel syndrome (yiangou 2001, Chan 2003). Perivous study has shown that nerve

injure leads to immnunoreactivity changes from trpv1 negative to positive in large

diameter neurons after nerve injury(Hudson 2001). A similar gain of capsaicin sensitivity

was seen in calcium imaging recently after nerve injury (Kim 2014 neuron). These

phenotypic switch was mostly seen in nerve injury and chronic pain(Costigan M 2009

Annu Rev Neurosci). Interestingly, cancer pain also leads to increase of trpv1

expression in both myelinated DRG neurons and perptidergic unmyelinated DRG

neurons (Niiyama Y, 2007 neuroscience). In our experiment, the intracellular calcium

response to capsaicin is also enhanced in terms of percentages of positive cells. The

percentage of TRPV1 responsive DRG rose in pain group was higher compared with

control group. The percentage change might reflect a phenotype change in DRG

population. This findings extended previous research results by showing that

unregulated trpv1 responsiveness in primary-afferent fibers occurs in postoperative

pain,

Together, these data are consistent with the hypothesis that the TRPV1 contribute to

postoperative pain DRG excitability. Indeed, the responses are both elevated in pod1

and pod7 when stimulated with capsaicin. To further understand the role to trpv1-

positive neurons in development of postoperative pain, we studied the Pod7 response in

DRGS. On PoD7, We found the the amplitude is reduced and d the percentage is

recovered. The amplitudes of capsaicin evoked calcium transients and percentages of

capsaicin responsive neurons are not higher in surgery group when compared with

control. This showed that the surgery induced a recoverable enhanced response in

DRGS indicating the disappearance of the inflammaotory part of postoperative pain.

However on pod7 the duration of capsaicin induced calcium response remains longer

in surgery group. This could due to the long- lasting remaining sensitization of DRGS

after surgery. This extended effect of surgery on calcium responses was reflected by

our wheel running results, which showed that on Pod7 there is still a difference between

RTX and no treatment group in running distance(On pod6 and pod7 there is a sig diff

between RTX and no drug laminectomy in running distance (two way anova, for multiple

t- test, day 3,4,5,6,7 is different. P=0.016, 0.0013,0.0013,0.0002,0.0004).

In our study, One day after the laminectomy, there was also a significant increase in the

magnitude and decrease in the decay of potassium evoked calcium transients in DiI

labeled neurons. We found the response duration, amplitude of DRG response to k30

to be elevated on POD1. And this enhanced response disappeared on POD7. Evidence

showed that the nerve injury in DRG leads to decrease in resting calcium concentration

(Fuchs ,2005) and a decrease in both the magnitude and decay of evoked calcium

transients (Fuchs 2007)and nerve injury also leads to a decreased voltage gated

calcium currents in injured DRG (MaCallum 2003, Baccei and Kocsis,2000). Also there

was reports that inflammation is associated with increases in magnitude and decrease

in decay of the high potassium evoked transient in small putative nociceptive

afferents(Lu 2008,neuroscience). In the present postoperative pain model, high

potassium induced weak excitation of naïve DRG , but strongly activate the DRG in

laminectomy group as indicated by increase in calcium transients’ amplitudes and

durations. This augmented responsiveness indicated neuronal sensitization. This

stronger activation is possibly due to trpv1 sensitization in laminectomy group because

inflammatory mediators induces an acute sensitization of nociceptive DRG neurons

partially through sensitization of the TRPV1(Zhu W, 2007 mol cell neurosci). The high

responsiveness to KCl and capsaicin shown in our postoperative pain model support

the idea of sensitization of DRG and phenotypic changes in DRGs after surgery. This

enhanced responsiveness to potassium disappeared on POD7.

Using this pain model, we also showed that the significantly augmented DRG neurons’

response to both potassium and capsaicin challenges are blunted by the application of

TPV1 agonist RTX. Also the percentage changes happened following the surgery are

also prevented by pretreatment of RTX. RTX mediated antinociceptive effects both in

home cage locomotion and wheel running. Trpv1 agonists-induced analgesia is

proposed be due to receptor desensitization because of a sustained activation of

receptors()Jeffry Jap los one 2009, Raisinghani M 2005 j physiol). I. t. RTX couls cause

aa sustained increased freuqencey of spontaneous EPSCs and lead to a

desensitization response and depolarization block, synaptic failures(Jeffry Ja plosone

2009 sikand p, 2007 j physiol).. RTX has been shown to cause both spinal cord and

DRG changes if applied intrathecal.

RTX could induce a degenaeraion of trpv1 positive neurons by causing a neurotoxic

influx of calcium ion through the channels resulting a calcium induced neuronal

death(karai 2004). Our data extended the previous findings by showing that the

percentage of DRG neurons responveis to capsicien is significantly reduced aftet RTX

treatment. Also it has been shown that the ablation of central terminals of trv1

expressing neurons occured. after i.t. RTX (Bishinoi M 2011 j pain). Even when used

perineurally and remote from the neuronal perikary in DRG , RTX still could ablate DRG

TRPV1 positive neurons (Neubert 2008). RTX is neuroexcitotoxic and ablate neurons.

RTX is a promising drug for treatment because of its desensitizing effect and ablate

effect. Single dose might desensitize ,repeated dose or high dose could cause

neurotoxity and permanently degenerate nerve Trpvv1 + nerve endings (Ladarola

2011). RTX induced a prolonged increase in intracellular calcium in Trvp1 positive

neurons in human dorsal root ganglion cultures, , while leaving other adjacent neurons

unaffected(Karai, 2004). It has been proved that intrathecal RTX could reduce TRpv1,

CGRP and trpa1 immunoreactivities in the lumbar dorsal root ganglia and (karai 2004,

Neubert 2008 Klafke 2012 neuroscience), indicating the deltetion ot DRG TRPV1-

expresssing neurons(Neubert). Intrathecal rtx also produce ablation of central nerve

terminals of DRG neurons indicated by decreased immune reaction of TRV1 in spinal

cord dorsal horn (Bishnoi 2011). This cellular changes were reflectd in behavioral tests

such as prolonged hind paw thermal stimulation latency and carrageenan-induced

inflammatory therma lanalgesia(Bishnoi 2011). Our thermal test on hind paw confirmed

the prolonged radiant noxiouls thermal stimulus latency and suggested the specific

deletion of trpv1 positive neurons. In the present study, intrathecal RTX reduced the

calcium response. We found that RTX producing a significantly reduced responsiveness

to potassium and capsacin in DRGs.Also In calcium imaging, the percentages of trpv1

responsive DRG neurons decrease after RTX application both in postoperative rats and

sham rats further proved that the intrathecal RTX targeted trpv1 positive neurons in

DRG and made them inert to high potassium and capsaicin. We propose that i.t. RTX

also affect DRG trpv1 channels because RTX diffused to DRG and act on primary

afferent neurons. This may partly explained our finding that after rtx, the DRG response

to capsaicin are reduced.

This further extended the findings that RTX decreasing the TRPV1 immunoreactivities

in DRG after both perineural and intrathecal RTX application(neubert Jk, 2008, Klafke

2012 neuroscience)

RTX has been used for debilitating arthritic and cancer pain . In rats of postoperative

pain, RTX can significantly reduce the pain(my paper). Also the RTX has been shown to

be effective in treatment of neuropathic pain(gariel Tender, spinaljournal 2008) because

RTX jinjection acuse an excitotoxic effect when injected into sensory ganglia. RTX is a

transient receptor potential vanilloid 1 channel agonist. It also contributes to

inflammatory and heat allodynia relieve. Our intrathecal injection of RTX also cause an

inert DRG neuron reaction to K30 and capsaicin, both on pod1 and pod7. Extensive

research dta have shown that trpV1 positive neurons mediate thermal hyperalgesia in

pain models.(11-16 of Tender 2008 spine ournal). Moreover , the same cells apppeart

to be responsible ofre the mediation of neurogenic inflamesion(13.17-19), blockage of

trpv1 apears eefective in reversing established hyperalgesia associate with

inflammation(18). Our data corroborated that RTX could abolish the heat induce pain in

rats, evidence by the extenede time response time to the heat application on hindpaw in

rats treated with i.t. RTX. The paw became less responsive to heat. These results are in

concordance with previous studies showing theat trpv1-posticitve neurons mediated

termal hyperalgesia.

Also it is shown that inhibition of trpv1 centrally could inhibit the mechanical

hyperalgesia even it is not a mechanotransducer(Kim 2014 Neuron). So far there have

been no data to demonstrate the involvement of trpv1-positive neurons in mechanical

sensitivity or allodynia. However, some earlier reports showed that RTX injected in

DRGs could elevated the average withdrawal threshold of the paws and the magnitude

of touch –evoked allodynia was improved, which means the RTX improved tactile

allodynia via selectively eliminating the trv1-positive cells in the DRGs. This could be

explained by the cross-talk between A-beta, A-delta and C-fiber neurons in DRGs (23 of

tender 2008) . After operation, large amounts of glutamate and aspartate are released

into extracellular space and the inhibitory neurotransmitter GABA is reduced in spinal

dorsal horn (22) and also in the DRGs, resulting the hypersensitivity of A fibers and C

fibers. When use a touch test of allodynia, the A-beta fibers carried the signals to the

DRG and excited not only the A-beta cell bodies but also A-delta and C neurons

because of the cross-excitation. The excitation of a A-delta and C neurons subsequently

contribute to the touch induced pain and personal’s experience and emotional status.

Thus, deletion of trpv1 neurons (include A-delta and C fibers) could reduce the

excitation of A-delta and C- fibers in DRGs and thus reduce the whole animal’s

response to touch.

Also even we did not test the mechanical threshold of back region. There was evidence

showing that the surgery affected the cutaneous sensitivity in the back region after

thoracotomy and RTX could relieve the allodynia developed after(Shin 2010) surgery11.

It can relieve pain like shown in behavioral tests wheel running and spontaneous

activities. It already has been shown when used in large doses intrathecally(3.8ug/kg), it

is able to alleviate capsaicin induced nocifensive behavior(bishnoi 2011 j pain) and both

capsaicin and carrageenan induced inflame thermal hypersensitivity  (bishnoi 2011 j

pain).    Also it is shown that inhibition of trpv1 centrally could inhibit the mechanical

hyperalgesia even it is not a mechanotransducer(Kim 2014 Neuron).

We observed the spontaneous activity after surgery and the wheel running behavior

after laminectomy instead, it is shown that the wheel running and home cage activity are

changed by laminectomy and recovered partially due to RTX. We say the RTX

improved the running distance and the spontaneous activity in postoperative rats. Such

as rearing and fine movements in homecage, which proved that RTX mediated

antinociceptive effects and even after intrathecal RTX, animal maintain the motor

control. There was report that RTX caused a long lasting analgesia in cancer pain

model even up to 14 week after application and the hind paw heat response was

significantly reduced (Brown 2005). Our heat test further confirmed that intrathecal RTX

induced a higher heat response threshold. Also it has been shown that lumbar epidural

application of RTX resulted a focused distribution to lumbar neurons and lead to a

profound and segmental analgesia12. Recently it was shown that intrathecal RTX could

lead to dose-dependent deletion of TRPV1 CGRP and phosphor ERK

immunoreactivities in superficial spinal cord such as laminae I and II. Other studies

showed the physiological sensation could recover 2 weeks after RTx treatment2.

In summary, the high responsiveness to capsaicin and potassium in DRG reflected the

sensitization of neurons and this indicates high excitability and more neurotransmitter

release in peripheral nociceptive neurons. RTX is a promising pain killer because both

the calicium imaging and behavioral test support its use in postoperative pain.

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