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Jan 2019

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Minimally Invasive Oral Surgery Induction of the FRICT-ION Chronic Neuropathic Pain Model
微创口腔手术诱导FRICT-ION慢性神经病理性疼痛模型的建立   

Marena A. MonteraMarena A. Montera*Karin N. WestlundKarin N. Westlund*  (*共同第一作者)
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Abstract

An easily induced preclinical trigeminal neuropathic nerve injury model is described here for the study of chronic pain, the model acronym FRICT-ION (Foramen Rotundum Inflammatory Constriction Trigeminal InfraOrbital Nerve). In patients, neuropathic pain is thought to be related to vascular alignment or multiple sclerosis along this small trigeminal nerve branch (V2) innervating the maxillary teeth and middle third of the face. With no detectable outward physical signs, the FRICT-ION model is ideal for blinded studies. The acronym FRICT-ION applied relates to the persistence of the trigeminal neuropathic pain model likely due to sliding irritation with normal chewing in the mice. A step-by-step method to induce the mild chronic rodent neuropathic pain model is described here. The surgery is performed orally through a tiny surgical slit inside the cheek crease to align a chromic gut suture irritant along the nerve as it passes into the skull. The model allows testing of non-evoked subjective measures and evoked quantitative mechanical hypersensitivity (allodynia) testing with von Frey filaments through at least 10-14 weeks (100 days). Anxiety and depression behaviors develop within 3-6 weeks relevant to the affective component of chronic pain. While many pain drugs have failed based on testing performed in the acute animal models available, the more stable and easily replicated trigeminal inflammatory compression model is the better suited for understanding both mechanistic and affective components of nerve injury-induced chronic neuropathic pain states as well as the more ideal for preclinical trials of novel non-opioid pain relief therapies.

Keywords: Chronic pain (慢性疼痛), Orofacial pain (口面疼痛), Neuropathic pain (神经性疼痛), Neuralgia (神经痛), Chromic gut (铬肠线), Infraorbital nerve (眶下神经), Anxiety (焦虑)

Background

Damaged peripheral nerves cause persisting overactivation referred to as “neuropathic pain”. A serious consequence of persisting nerve injury pain is the transition to chronic pain. Neuropathic pain is the result of changes in the signaling molecules in the peripheral nerve cells that eventually cause central sensitization and changes in the brain’s pain and affective circuitry. A major obstacle to better understanding of pathophysiological mechanisms of chronic neuropathic pain and development of effective therapeutics is lack of available experimental animal models that mimic established chronic pain. To emulate chronic orofacial neuropathic pain, the FRICT-ION (Foramen Rotundum Inflammatory Constriction of Trigeminal InfraOrbital Nerve) nerve injury model was developed in mice to study behavioral, pharmacological, cellular, and molecular mechanisms persisting during chronic neuropathic pain.

While many nerve injury models have been developed that allow study of nociceptive mechanisms and pain-related behaviors in the short term, the resilience of natural healing processes typically reverses nerve injury models within 3-4 weeks in neuropathic pain models. The method described below is one of the few models that allow long-term studies suitable for better understanding of clinical pain states and for testing experimental therapies. Several innate differences in the trigeminal nerve are likely responsible for the chronic persistence of the neuropathic model. The great importance of the trigeminal nerve to survival is postulated to be one characteristic responsible for the ease in producing persistence of trigeminal nerve injury pain and its effects on psychological well-being (Carlson, 2007). Anatomical proximity to the brain and the unique circuitry of the trigeminal system with one less synapse forming more direct connectivity with the limbic circuitry are also likely responsible for the intensity and persistence of neuropathic pain associated with the trigeminal nerve (Rodriguez et al., 2017).

Previous successful chronic trigeminal pain models described have been induced by loose chromic gut suture tie of the infraorbital nerve (ION) in rats (CCI-ION) behind the eye (Vos et al., 1994; Kniffin et al., 2015) or on the snout in rats (dIoN-CCI) and mice (DIONI) (Ding et al., 2017; Hardt et al., 2019) (Figure 1). Alternatively, the intraoral approach has also been used to induce the CCI-ION model in rats (Immamura et al., 1997). We previously introduced a trigeminal nerve hypersensitivity model induced by sliding the irritative chromic gut suture into the ION fissure behind the eye in mice creating a trigeminal inflammatory compression (TIC) (Figure 1) (Ma et al., 2012 and 2015; Lyons et al., 2015 and 2018). The surgical approach to the ION behind the eye is technically challenging and the vasculature prone to bleeding, often severely. After induction of trigeminal nerve injury models, mechanical hypersensitivity develops within a week and persists indefinitely in the ION’s whisker pad receptive field. After trigeminal nerve injury, cognitive deficits are reported within 3 weeks in rats and anxiety- and depression-related symptoms emerge in subsequent weeks after injury (>6 weeks) in rodents indicating brain origin and potential circuitry neuroplasticity within brain regions are responsible for these behaviors accompanying chronic pain (Yalcin et al., 2011 and 2014; Kniffin et al., 2015; Lyons et al., 2015 and 2018). Estimates are that in weeks 6-8 post-surgery, mice have experienced pain equivalent to 6-8 human years and persistence of models in this time frame can easily be considered chronic (Dutta and Sengupta, 2016; Hannaman et al., 2016). The interplay between psychologic and physical functioning is a particular consideration in patients with orofacial pain (Carlson, 2007).


Figure 1. Surgical sites utilized in trigeminal neuropathic pain models. 1. TIC, 2. CCI-ION, 3. FRICT-ION, 4. dIoN-CCI and DIONI.

Better models for the study of chronic neuropathic pain are needed to discover more effective treatments since the current treatment of choice, microvascular decompression surgery, in patients with chronic trigeminal nerve pain or multiple sclerosis pain wanes over time with reoccurrence typically at 3 years (Xia et al., 2014). The pain-free rate at 5 and 10 years decreases to 61% and 44% after decompression surgery to place a Teflon spacer and after stereotaxic radioablation is only 47%, and 27%, respectively (Wang et al., 2018). Teflon granulomas are reported in 5.6% of patients after microvascular decompression (Chen et al., 2000).

The FRICT-ION method provides a simpler intraoral approach (Figure 1). The benefits of using the mouse FRICT-ION model of trigeminal nerve injury for study of chronic neuropathic pain in general are many. The model induces long-term chronic neuropathic pain with no other apparent side effects or outward physical indication. The mouse experiences no health or weight gain issues. The method exposes the rodent to only minutes of isoflurane anesthesia but sensitivity in the receptive field on the whiskerpad persists indefinitely. If done correctly, the minimally invasive surgery has little or no bleeding. The model is quickly and easily mastered following the instructions provided below.

Surgical Induction of the FRICT-ION Model
We induce the model in either BALBc or C57Bl/6 mice (20 to 25 g; 8-10 weeks; Harlan Laboratories, Indianapolis, IN). We have performed the procedure in older mice with no complications. The surgery is performed open mouth through a tiny slit to align chromic gut suture along the trigeminal nerve. The constant but minimal nerve irritation likely produced with chewing is similar to one known cause of trigeminal neuropathic pain, irritation by proximity of the nerve to pulsating brainstem vasculature which is relieved by insertion of a Teflon sheet (Dandy, 1934; Xia et al., 2014). The extended duration of orofacial neuropathic pain models provides an optimal platform for preclinical testing of potential therapeutics since mechanical and cold sensitivities, anxiety, and depression-like behaviors are measurable.

The model illustrated here is induced by inserting 3 mm of chromic gut suture intraorally into the tight space where the infraorbital nerve (ION) passes through the bony infraorbital foramen. Similar to nerve biopsies from patients (Weis et al., 2012), the TIC model we reported previously using the intraorbital surgical approach did not cause the axonal degeneration seen in tied or cut nerve models (Ma et al., 2012). As a result, the TIC model induced hypersensitivities similar to those in the human condition, i.e., mechanical and cold allodynia, but not heat hypersensitivity, along with anxiety and depression. Though not tested as yet, we are hopeful that these results will be duplicated using the intraoral approach to induce the FRICT-ION model. The model is illustrated here induced on the right side of the mice in all figures. The model has been induced in both males and females, BALBc and C57Bl/6, and while not yet tested by our lab, likely could also be done in rats. The intraoral approach has been used previously in rats to induce the nerve tied CCI-ION model (Immamura et al., 1997).

Materials and Reagents

  1. 0.2 cm diameter corroborated evac tubing (Patterson Veterinary, catalog number: 07-8914311 )
  2. 1 cm diameter Tygon tube (Fisher Scientific, catalog number: 14-171-104 )
  3. Scavenger filter of the anesthesia unit (Patterson Scientific WAG unit, catalog number: 78909457 )
  4. Stabilization/tension ties constructed from rubber bands and surgical silk (shopmedvet.com, 5-0 silk; 100M cassette SKU: SLK50)
  5. Embroidery floss (DMC, catalog number: 117S-3380 )
  6. Binder clip, ACCO 2” (Staples, catalog number: 72100 )
  7. Cotton-tipped applicators (shopmedvet.com, 6”, non-sterile, SKU: CTA6)
  8. Gloves
  9. BALBc or C57Bl/6 mice (20 to 25 g; 8-10 weeks; Harlan Laboratories, Indianapolis, IN)
  10. 70% ethanol (local source)
  11. Sucrose

Equipment

  1. 4-0 chromic gut suture (cut into 3 mm lengths) (Ethicon, catalog number: 635H )
  2. Forceps (Dumont No. 5, Fine Science Tools, catalog number: 11251-10 ; Graefe Angled Serrated, Fine Science Tools, catalog number: 11049-10 )
  3. No. 11 scalpel blades (Feather/Electron Microscopy #11, catalog number: 72044-11 , VWR, catalog number: 102097-822 )
  4. Scalpel handle #3 (Walter Stern 320-051) (VWR, catalog number: 25607-947 )
  5. Bead sterilizer (Germinator 500 Glass Bead Sterilizer, Cell Point Scientific 5-1450) (VWR, catalog number: 101326-488 )
  6. Heating pad/recovery station for mice (HTP-1500 Adroit Veterinary Heat Pump and Soft Temp Veterinary Heat Pad V016) (Patterson Veterinary Supply, catalog numbers: 78910655 and 78910866 )
  7. Surgery setup (see Procedure section below)
  8. Dissecting scope (Optika SZN-10 Trinocular Stereo Zoom Microscope on Hinged Overhanging Stand, New York Microscope Company, catalog number: OPSZN-10 )
  9. Isoflurane anesthesia machine [Tec 3 EX Isoflurane Standard Fill Vaporizer, Versa II Anesthesia system, with Mapleson-D Non-Rebreathing (NRB) System] (Patterson Veterinary Supply, catalog numbers: 78703592 , 78910545 with 78909636 )
  10. Base Plate, S&T Ball Chain Retraction (Fine Science Tools, catalog number: 18100-00 )
  11. Fiber Optic Light (Dolan-Jenner Fiber-lite Illumination System 181-1) (VWR, catalog number: 41446-062 )
  12. Von Frey Filaments (Semmes-Weinstein, 20 monofilaments, BioSeb, bioseb.com)
  13. Autoclave

Software

  1. Ethovision XT8 videotracking system software (Noldus Information Technology, Leesburg, VA, USA) for analysis of movement in the affective behavioral tests
  2. GraphPad Prism 8 for statistical analysis and data presentation (GraphPad Software Inc., La Jolla, CA)

Procedure

  1. The surgery is performed on an alcohol cleaned flat surface beneath a dissection scope with an apparatus mounted with stabilization restraints to hold the mouth open. Recommendation is to use an S&T Ball Chain Retraction System Base Plate. Two lengths of 5-0 surgical silk (or embroidery thread, soft string, to avoid damage to the mouse’s tongue) with rubber bands knotted at the strings’ ends can be hooked to the plate’s ribbed edges. The surgical silk is drawn tight across the mouse’s mouth for restraint of the mouth in an open position for anesthesia and surgery (Figure 2). Place the heating pad recovery station on a table adjacent to the surgery setup so that mice can be quickly transferred to the station upon completion of surgery. The station should be set to 41 degrees Celsius. We place an empty housing cage on top of the heating pad about an hour prior to the surgeries to allow the cage to heat up to a comfortable temperature; half on the pad, half off.


    Figure 2. Mouse anesthesia setup

  2. Since this is an oral surgery, a setup is needed that will reliably deliver isoflurane to the mouse while still leaving the mouth free for the operation. Recommendation is to use a two-tube system: a 0.2 cm diameter tube to deliver the anesthesia, slid inside a larger 1 cm diameter Tygon tube. The outer tube will connect to the scavenger filter of the anesthesia unit (Figure 2). Figure 3 provides further clarification of the orientation to accommodate the mouse’s nose. There should be an 8 mm gap between the tip of the inner tube and the tip of the outer tube. The mouse’s nose can be slid into the outer tube to allow anesthesia delivery, while still allowing the mouth to be opened and manipulated during surgery. The procedure is done under a dissection scope at a magnification of 10x, though higher magnifications might be beneficial when first learning the procedure.


    Figure 3. Mouse restraint

  3. Sterilize surgery tools (scalpel handle, forceps) in a bead sterilizer, autoclave, or spray them with 70% ethanol and air dry.
  4. Set up the anesthesia machine with a splitter to provide dual tubing approach of the mixed oxygen/isoflurane to both the induction chamber and the surgical setup. Optimal settings will depend on the isoflurane delivery vaporizer cannister, but current recommendation is an oxygen level of 1.5 and an isoflurane level of 3%.
  5. Place the mouse into the induction chamber for initial anesthesia induction; the typical rapid breathing will begin to slow down to deeper breaths, moving from the upper chest to the lower chest. This will take about one to two minutes, depending on the mouse and the chamber size.
  6. Switch anesthetic flow to the surgical area tubing.
  7. Move the anesthetized mouse quickly to the surgical area beneath the dissection scope placing its nose into the Tygon tubing to maintain anesthesia depth and to avoid having to replace the mouse back into the induction chamber to repeat the anesthesia process. Should any movement occur, place the mouse back into the induction chamber and repeat this process. Movement of any kind by the mouse indicates the tubing is not providing adequate isoflurane flow.
  8. Restrain the mouse for surgery. Hook one restraint under the upper two front teeth, and the other restraint underneath the lower two front teeth (Figure 4), gently pulling the mouth as wide open as possible while still keeping the mouse’s nose inside the anesthesia tubing. If necessary, use tape across the tail to help hold the mouse steady.


    Figure 4. Top-down view of open-mouth positioning

  9. Adjust the microscope and lighting as needed in order to have a clear visual into the mouse’s mouth (Figure 5). From here, begin the procedure.


    Figure 5. Broad view of the surgery and restraint set-up

  10. Using a scalpel blade (#11) mounted on a surgical scalpel handle (#3), make a small incision into the mouse’s lip inner bucchal margin crease a little below the roof of the mouth (Figure 6).


    Figure 6. Highlighted surgical site provides illustration of the 2 mm incision (15x). The diagram reveals the underlying trigeminal nerve branches.

  11. Gently cut away the soft tissue using the tip of the scalpel blade. You will eventually create a hole to reveal the trigeminal maxillary infraorbital nerve branches that innervate the teeth and whiskerpad coming together posteriorly at the bony foramen rotundum to enter the skull (Figure 7). Initially opening this site a little wider in a cadaver or practice mouse will allow visualization of the infraorbital nerve innervation and the foramen rotundum where the chromic gut will be inserted (Figures 8 and 9). The surgical site ordinarily should be quite small; no more than a couple of millimeters, and no suturing of the wound should be needed if an experienced experimenter did the surgery. Please note that the dissection and surgery have been exaggerated in all of the descriptive images provided. It is unlikely, but there would be excessive bleeding from the wound if the cut is deeper than intended, then stop the procedure and do not use the mouse for further experimentation.


    Figure 7. Demonstration of nerve bundle entering into foramen. The cut is exaggerated for better display in this higher power view (15x).


    Figure 8. Demonstration of chromic gut suture placement within foramen exaggerated dissection (15x)


    Figure 9. Insertion of chromic gut suture into the foramen rotundum alongside the ION nerve (15x)

  12. Using small forceps (recommended: Dumont #5), grip 3 mm suture by the end and angle it into the hole you have just cut in the crease of the mouse’s cheek (Figure 8). Then slide the suture into the foramen, alongside the ION. The suture should go in with very little resistance; if you feel resistance, it means that you do not have the right angle and are not guiding the suture into the foramen. The nerve should never be touched with the metal surgical instruments.
  13. Push the suture into the foramen as far as possible with the forceps (Figures 8 and 9). Then, gently let go, and use the closed tips of your forceps to push the end of the suture further through the foramen. You should just barely be able to see the end of the suture if you have pushed it in correctly.
  14. The incision used in this model is small enough that no sutures are required to close the wound. In both shams and the FRICT-ION model, the wound begins to close within the first twenty-four hours (Figure 10).


    Figure 10. Sham Surgery at 0, 24, and 48 h post surgery. Wound is mostly closed within 24 h and is completely closed by 48 h. Only slight vascularity can be seen at 24 h.

  15. Place the mouse into the heat recovery station until it awakes from the anesthesia and is mobile before returning to the home cage. Mice should be checked an hour after surgery to ensure all normal motor function has returned. (This is a precaution, as typically mice are fully functional within minutes of removal from anesthesia.) Mice should then be checked daily for the week following surgery, including assessing weights and surface glances into the mouth to ensure no exacerbation of the wound or infection has occurred. It is likely that mice will not gain weight the day following the surgery, but by day two should be back to the typical gradual weight gain.
      There should be no noticeable change in behavior; FRICT-ION mice should appear similar to naïve mice to the naked eye. If there is a change in behavior, such as weight loss, lethargy, etc., do not use the mouse for further experimentation. If the weight falls below eighty percent of the mouse’s weight prior to surgery, the mouse should be checked by veterinary staff and, if applicable, humanely euthanized.

Data analysis

Nociceptive and anxiety related behavioral testing and data analysis
Animals are moved from the housing room and acclimated in the testing room for 30 min in their home cages prior to testing. Animals are acclimated to gentle restraint (held in gloved hand for whisker pad tests) to assess sensitivity to mechanical stimulation applied to the whiskerpad, i.e., reflexive withdrawal. After 10 training sessions to acclimate the rodent to being held in a gloved hand, sensitivity of the face to mechanical is quantified by the number of withdrawal events from graded thin nylon von Frey filaments with defined bending forces (tensile strength). Stimulation with the lowest fiber (0.008 g, 1.65) is not detectable on the back of the human hand; the largest fiber (6.0 g, 4.74) provides the sensation of a blunt paper clip wire end. Animals are free to voluntarily move their head away from the stimulus. A single trial consists of 5 applications of von Frey filaments beginning mid-range (0.4 g, 3.61) applied once every 3 to 4 s. If a positive response occurs, the next weaker filament is applied instead, continuing until there is no response; if no positive response is evoked, the next stronger filament is applied and continues until there is a response (Chaplan et al., 1994). The mean occurrence of withdrawal events in each of the trials is expressed as the number of responses out of 5, 0 indicates no withdrawal, and 5 indicates the maximum number of withdrawals. Responses to decreased gram force compared to controls indicate increased sensitivity. After induction of trigeminal nerve injury models, statistically significant mechanical hypersensitivity develops reliably within a week and persists indefinitely as tested with calibrated von Frey filaments in the ION’s whisker pad receptive field (Figure 11). The mechanical stimulation threshold on both the ipsilateral and contralateral whisker pads is significantly decreased in mice after the surgery compared to surgical sham and control mice. This decrease persists until euthanasia, ten weeks post-surgery.


Figure 11. Male mice were tested for mechanical allodynia on the whisker pad using von Frey filaments. Two-way ANOVA (Dunnett’s multiple comparisons test) was performed on these data: n = 5 per group, * indicates a P-value of < 0.05 [Surgery side: F(20, 198) = 9.178, Contralateral side: F(22, 144) = 32.20]. In post-hoc analyses, Bonferroni adjustment to all P-values for week-by-week comparisons of FRICT-ION versus Control yields all nine P-values < 0.0009.

While management of neuropathic pain is important because in many cases it is severe and unrelenting, study to develop better treatment of the anxiety and depression comorbidities is also of prime importance in overall quality of life (Moulin et al., 2007). Another major advantage of the FRICT-ION model is the ability to examine these behaviors at chronic time points. Testable in the model are anxiety-related measures such as zero maze, light-dark preference, and open field exploratory behaviors. In Figure 12, male mice were placed into a light/dark chamber and monitored for ten minutes. In this test, one chamber was lighted with a bright bulb while the other was kept dark, and mice were allowed free access to either chamber. Mice with FRICT-ION surgery were significantly less likely to spend time in the light chamber, and explored the chamber less in general, which is indicative of anxiety.


Figure 12. Anxiety behaviors shown were tested in week 8 post model induction (n = 5 per group, * indicates a P-value of < 0.05, comparisons run using a t-test).

Both cognitive and the sucrose splash depression test (Figure 13) can be utilized to differentiate these higher order consequences of chronic pain in the FRICT-ION model (Yalcin et al., 2011; Kniffin et al., 2015). Mice were given the sucrose splash test, in which 30% sucrose water is sprayed onto mouse’s rump, and mice were monitored for five minutes to determine the number of times they groomed the rump and cage area wetted. Mice with FRICT-ION surgery groomed significantly less often, which is indicative of depression behavior.


Figure 13. Depression behaviors were tested in week 8 post model induction in male mice (n = 5 per group, * indicates a P-value of < 0.05, comparisons run using a t-test).

Notes

  1. For the sham surgery, the small cut is made in the bucchal margin but no suture inserted. If done correctly, this process should not induce any bleeding inside the mouse’s mouth or brain cavity. Remove the mouse from its restraints and place on a paper towel in a clean cage with bedding that has been sitting on a heating pad. The mouse should become conscious less than a minute after you have removed it from anesthesia, and should be up and walking normally within five minutes. The mouse should recover from this surgery with ease, and be back to normal behavior by the end of the day.
  2. With practice, this procedure should take no more than a few minutes. It is important to perform this procedure quickly to avoid the side effects of long-term exposure to anesthesia.
  3. This procedure should be done with no bleeding; however, if bleeding occurs, simply apply pressure to the site with a cotton swab until the bleeding subsides.
  4. Mice can be housed together, and should not require any care after surgery except for standard monitoring.

Acknowledgments

The authors wish to acknowledge the laboratory management skills of Katherine Gott. Departmental funds and NIH grant R21 DE028096 support the Anesthesiology Department Pain Laboratory. The minimally invasive intraoral FRICT-ION approach described here was first used in a recent publication, but we referred to it there as the “TIC” method (Zhang et al., 2019). The FRICT-ION protocol was adapted or modified from our previous trigeminal inflammatory compression (TIC) method where chromic gut suture was aligned along the infraorbital nerve in mice using the more invasive surgical approach behind the eye (Ma et al., 2012 and 2015; Lyons et al., 2015 and 2018). The authors acknowledge the insight provided by former research scientist, Dr. Fei Ma, who introduced the concept of inducing infraorbital nerve compression by suture insertion into a foramen rather than tying the nerve to produce a mouse preclinical model of chronic trigeminal neuropathic pain.

Competing interests

KNW is an unpaid advisor to USA Elixiria Biotech Inc. and a Research Physiologist at the Albuquerque VA Health Care System. This communication does not necessarily reflect the views of the Department of Veterans Affairs or the U.S. government.

Ethics

All studies were designed to minimize animal use and performed in accordance with institutional approvals and in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996.

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简介

[摘要 ] 一种容易诱发临床前三叉神经性神经损伤模型中描述了在此对于研究的慢性疼痛,Ť 他模型缩写FRICT-ION (˚F Oramen ř Otundum 我Nflammatory Ç Onstriction Ť Rigeminal 我NFRA ö Rbital Ñ ERVE),在患者,神经性疼痛被认为与支配上颌牙齿和中三分之一o的小三叉神经分支(V2)上的血管排列或多发性硬化有关 F中的人脸。没有检测到对外体征,该FRICT-ION模式非常适合盲法研究。缩写FRICT-ION应用RELA 维护设备到持久性的三叉神经性疼痛模型可能是由于滑有刺激性,一般正常的咀嚼中的小鼠,一种步骤一步的方法诱导的慢性轻度的啮齿动物神经疼痛模型描述了这里。在手术进行口服通过一个很小的手术狭缝处的脸颊折痕对齐铬肠线缝合刺激沿神经,因为它穿入该模型允许使用von Frey细丝测试至少10-14周(100天)的非诱发性主观措施和诱发性定量机械性超敏反应(异常性疼痛),在与焦虑相关的3-6周内会出现焦虑和抑郁行为尽管根据在可用的急性动物模型中进行的测试,许多止痛药均以失败告终,但更稳定,更容易复制的三叉神经炎。压缩模式保守党是是更好的适合了解这两种机理而Affec 略去组件的神经伤病引致诱发的慢性神经性疼痛状态以及在更理想的临床前试验的新型非阿片类止痛疗法。

[背景 ] 受损的周围神经导致持续过度激活,称为“神经性疼痛”,持续存在的神经损伤疼痛的严重后果是向慢性疼痛的转变,神经性疼痛是周围神经细胞信号分子变化的结果,最终导致中央宣传和变化在大脑中的痛苦和情感的电路。一个主要障碍以便更好地了解病理生理机制慢性神经性疼痛和开发有效治疗缺乏中可用的实验动物模型模拟建立慢性疼痛。模仿慢性口面部神经性疼痛,在小鼠中建立了FRICT-ION(三叉神经下眶神经的圆孔圆形炎性收缩)神经损伤模型,以研究在慢性神经性疼痛中持续存在的行为,药理,细胞和分子机制。

许多神经损伤虽然车型已经开发了允许研究伤害性机制和痛苦- 。相关行为在短期内,应变能力自然愈合通常处理逆转神经损伤模型中3-4周神经病理性疼痛模型所描述的方法下面是少数能够长期研究的模型之一,适合于更好地了解临床疼痛状态并测试实验疗法。三叉神经的先天差异可能是神经性模型慢性持久性的原因。生存神经被认为是导致三叉神经损伤疼痛持续产生及其对心理健康的影响的特征之一(Carlson,2007)。解剖学上接近大脑以及三叉神经系统的独特回路较少的突触形成与边缘电路更直接的连接也很可能 负责与三叉神经相关的神经性疼痛的强度和持续性(Rodriguez et al。,2017)。

先前描述的成功的慢性三叉神经痛模型是由眼后大鼠(CCI-ION)的眶下神经(ION)的松散的铬色肠线缝合线(Vos 等人,1994; Kniffin 等人,2015)或以上引起的大鼠(dIoN -CCI)和小鼠(DIONI)的口鼻部(Ding 等人,2017; Hardt 等人,2019)(图1)。此外,口内入路也已被用于诱导CCI-ION模型在大鼠中(Immamura 等,1997)。我们先前引入了三叉神经超敏模型,该模型是通过将刺激性的铬肠缝合线滑入小鼠眼后的ION裂缝中而产生的三叉神经炎性压缩(TIC)(图1)(Ma 等人,2012年和2015年; Lyons等人,2015年和2018年)。眼后ION的手术方法在技术上具有挑战性,脉管系统容易出血,通常严重发作。在诱发三叉神经损伤模型后,机械性超敏反应会在一周内发展并无限期持续。三叉神经损伤后,大鼠在三周内出现认知功能障碍,啮齿动物在随后的几周内(> 6周)出现焦虑和抑郁相关症状,表明大脑起源和潜在的回路神经可塑性大脑区域内的这些行为是造成慢性疼痛的原因(Yalcin 等,2011和2014;Kniffin 等,2015; Lyons 等,2015和2018)。估计是术后6-8周,经历过痛苦的小鼠相当于人类6-8岁及持久性模型在这段时间内可以当作慢性(杜塔和森古普塔,2016年Hannaman 。等,2016)的在。心理和身体机能之间的相互作用是口面部疼痛患者的一个特别考虑因素(Carlson,2007)。





C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1903065--1368 Karin Westlund High 856375 \ Figs jpg \图1.jpg

1.手术部位图已用在三叉神经性疼痛模型。1. TIC,2. CCI-ION,3. FRICT-ION,4. DION -CCI 和DIONI 。



由于目前选择的治疗方法是微血管减压手术,因此对于慢性三叉神经痛或多发性硬化症患者,随着时间的流逝逐渐消失,通常需要3年的复发,因此需要更好的研究慢性神经性疼痛的模型以发现更有效的治疗方法(Xia 等等人,2014年)。在减压手术后放置特氟龙垫片后5年和10年的无痛率分别降至61%和44%,而立体定向放射消融后的无痛率分别仅为47%和27%(Wang 等。,2018)。在微血管减压后有5.6%的患者报告了铁氟龙肉芽肿(Chen et al。,2000)。

FRICT-ION方法提供了一种更简单的口腔内方法(图1)。使用小鼠三叉神经损伤FRICT-ION模型研究一般的慢性神经性疼痛有很多好处,该模型可诱导长期慢性神经性疼痛没有其他明显的副作用或外在的物理指示。鼠标没有出现健康或体重增加的问题。该方法使啮齿动物仅接受数分钟的异氟醚麻醉,但在晶须板上的感受野中的敏感性无限期地保持。手术几乎没有出血或完全没有出血。该模型可按照以下说明快速轻松地掌握。



FRICT-ION模型的手术诱导

诱导模型我们在这两种BALBC 或者C57BL / 6小鼠(20至25g; 8-10周;哈伦实验室,印第安纳波利斯,IN)。我们已经进行的程序我ñ 老年小鼠无并发症的Th。Ë手术行开放嘴巴上的细小缝隙使铬色肠线沿着三叉神经对齐。咀嚼可能产生的恒定但最小的神经刺激类似于三叉神经性疼痛的一种已知原因,这种刺激是由于神经靠近搏动的脑干脉管系统而缓解的。 (花花公子,1934年;夏插入Teflon片上。等人。,2014)病程延长,口面神经病理性疼痛模型提供了一个最佳平台,临床前试验的潜在治疗由于机械和冷敏感性,焦虑和抑郁- 样行为是可测量的。

此处所示的模式l是通过将3 mm的铬色肠线口经口插入狭窄空间而引起的,在该空间中,眶下神经(ION)穿过骨下眶孔,类似于患者的神经活检(Weis 等,2012)。我们先前使用眶内手术方法报道的TIC模型并未引起在束缚或割断的神经模型中见到的轴突变性(Ma 等,2012)。结果,TIC模型引起的超敏反应与人类情况相似,即,机械和冷异常,但不加热过敏,伴随着焦虑和抑郁。虽然没有测试到目前为止,我们希望这些结果将被复制使用口内入路诱导FRICT-ION模型。该模型画报这里诱导该模型已在雄性和雌性BALBc 和C57Bl / 6中进行了诱导,尽管尚未由我们的实验室进行测试,但也有可能在大鼠中完成。在所有图中,小鼠的内部都在右侧。方法是口服之前使用的大鼠诱导CCI捆绑-ION模型(神经Immamura 等人。,1997)。

关键字:慢性疼痛, 口面疼痛, 神经性疼痛, 神经痛, 铬肠线, 眶下神经, 焦虑

材料和试剂


 


直径为0.2厘米的经确认的真空输送管(Patterson Veterinary,目录号:07-8914311)
直径1厘米的Tygon 管(Fisher Scientific,目录号:14-171-104)
麻醉装置的清除剂过滤器(Patterson Scientific WAG装置,目录号:78909457)
由橡皮筋和手术用丝绸制成的稳定/张力扎带(shopmedvet.com,5-0丝绸; 100M暗盒SKU:SLK50)
绣花线(DMC,目录号:117S-3380)
活页夹,ACCO 2英寸(订书钉,目录号:72100)
棉花- 涂药(Shopmedvet.Com,6”,非无菌,SKU:CTA6)
手套
BALBc 或C57Bl / 6小鼠(20至25 g; 8-10周;印第安纳州印第安纳波利斯的Harlan Laboratories)
70%乙醇(本地来源)
蔗糖
 


配套设备


 


4-0铬肠线(切成3毫米长)(Ethicon ,目录号:635H )。
镊子(Duemont No.5 ,精细科学工具,目录号:11251-10;Graefe 角锯齿,精细科学工具,目录号:11049-10)
11号手术刀刀片(羽毛/电子显微镜#11,目录号:72044-11,VWR,目录号:102097-822 )
手术刀#3(Walter Stern 320-051 )(VWR,目录号:25607-947)
珠子灭菌器(Germinator 500玻璃珠灭菌器,Cell Point Scientific 5-1450 )(VWR,目录号:101326-488)
垫加热/恢复站小鼠(HTP-1500 ADROIT兽医热泵和软温度兽医隔热垫V016 )(帕特森兽医供应,猫考勤号码小号:78910655和78910866)
手术设置(下面的“ see 程序”部分)
解剖范围(可折叠悬架上的Optika SZN-10 三目立体变焦显微镜,纽约显微镜公司,目录号:OPSZN-10)
异氟烷麻醉机[ Tec的3 EX异氟醚标准填充汽化器的Versa II麻醉系统,随着Mapleson -D非再呼吸(NRB)系统] (帕特森兽医供应,猫考勤号码小号:78703592,78910545随着78909636)
底板,S&T滚珠链缩回(精细科学工具,目录号:18100-00)
光纤灯(Dolan-Jenner光纤照明系统181-1 )(VWR ,目录号:41446-062)
冯· 弗雷丝(Semmes-Weinstein,20条单丝,BioSeb ,bioseb.com)
高压釜
 


软体类


 


XT8 Ethovision Videotracking 系统软件(Noldus 信息技术,利斯堡,弗吉尼亚州,美国)对于分析中运动中的情感行为测试
图P 一d 棱镜8 对于统计分析和数据展示(的GraphPad 软件公司,拉拉霍亚,CA)
 


程序


 


该手术是演出在一个酒精清洗的平表面下方一个夹层范围有了一个设备安装有了稳定约束要举行的嘴打开。建议是要使用的小号安藤牛逼球链回缩系统基地板块。两个长度中5-0 手术丝(或绣花线,软字符串,为了避免伤害到的老鼠的舌头)用橡胶带打结在该串完能要带钩到该板块的其他罗纹边。该手术丝绸是拉紧对面的老鼠的嘴巴对于约束中的口在一个开放位置对于麻醉和手术(图2)。将在加热垫恢复站上一个表毗邻要在手术安装因此这小鼠可以被迅速转移到该站一旦完成中术。该站是否应要设定为41 度摄氏度。我们广场的空房屋凯奇在顶部中的加热垫关于一个小时之前到该外科手术要允许的凯奇为了热火最多要一个舒适的温度; 半在该垫,半关闭。
 


 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1903065--1368 Karin Westlund High 856375 \ Figs jpg \图2.jpg


图2.鼠标麻醉设置


 


由于这是一种口腔外科,一个设置是所需也就是说威尔可靠地交付异氟醚到该鼠标虽然仍留下的口免费为的操作。建议是要使用一个双- 管系统:一个0.2 厘米直径管要交付的麻醉,下跌内部甲更大的1 厘米直径的Tygon 管。该外管威尔连接到该清道夫过滤器中的麻醉单位(图2)。图3 提供了进一步澄清中的取向要迁就的老鼠的鼻子。有应成为一个8 毫米间隙之间的提示中的内管与该提示中的外管,该鼠标鼻子可以是下跌进入的外管要允许麻醉交货,虽然仍然允许的嘴巴要被打开而且操控下在外科杂志红霉素。该程序是完成在一个夹层范围在一个放大中10X,虽然较高的放大倍数可能会成为受益当首先学习的过程。
 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1903065--1368 Karin Westlund High 856375 \ Figs jpg \图3.jpg


图3. 鼠标约束


 


消毒手术工具(手术刀手柄,部队诗)在一个珠灭菌,高压灭菌器,或者喷涂他们用70 Pasento 乙醇和空气干燥。
设置最多的麻醉马茅根有了一个分路器要提供双管法中的混合氧气/异氟烷为了双方的感应商会与该手术安装程序。最佳设置将依靠在该异氟醚交货蒸发器储罐,但当前推荐通货膨胀是一个氧气级别中1.5 和一个异氟醚级别中3 Pasento。
放置在鼠标进入该感应商会对于初始麻醉诱导; 在典型的快速呼吸会开始要慢下来要更深层次的呼吸,移动从的上胸围为的下胸部。这将取关于一个要两分钟,根据在该鼠标与该箱体尺寸。
开关麻醉流程要在手术区管。
移动的麻醉鼠标快速为了将手术区域下方的解剖范围配售它的鼻子进入该的Tygon 管要保持麻醉深度而为了避免有要更换的鼠标返回进入该感应商会要重复的麻醉过程。是否应任何运动发生时,地方的鼠标返回进入该感应商会和重复此过程。运动中的任何一种通过将鼠标指示的油管是不是提供了足够的异氟醚流。
抑制的老鼠对于手术。胡克一个约束下的上两个前齿,而在其他约束下方的下两个前牙(图4),轻轻拉的嘴作为广开作为可能虽然仍保持在老鼠的鼻子里面的麻醉管。如果必要,使用磁带跨越的尾要帮助保持该鼠标稳定。
 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1903065--1368 Karin Westlund High 856375 \ Figs jpg \图4.jpg


图4.开口定位的俯视图


 


调整的显微镜和照明作为所需在订单要有无一个清晰的视觉进入的鼠标口(图5)。从这里,开始的过程。
 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1903065--1368 Karin Westlund High 856375 \ Figs jpg \图5.jpg


图5. 广泛的观中的手术和约束设置,向上


 


用一个手术刀刀片(#11)安装在一个外科手术刀手柄(#3),做一个小切口进入的老鼠的唇内Bucchal 保证金抗皱一个小下面的屋顶中的嘴(图6)。
 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1903065--1368 Karin Westlund High 856375 \ Figs jpg \图6.jpg


图6. 突出显示的外科网站提供插图中的2 毫米切口(15 X )。该图揭示的标的三叉神经分支。


 


轻轻地切离开的软组织使用的提示中的手术刀刀片。您将最终创建一个洞要显示的三叉上颌眶下神经分支那支配的牙齿和Whiskerpad 即将一起向后在该骨卵圆圆孔要输入的头骨(图7)。最初打开该网站一小更宽在一个尸体或者实践鼠标将允许可视化中的眶下神经支配而该卵圆圆孔哪里的艳色肠将要插入的(图8 和9)。该手术网站按说应该要相当小; 没有更比一夫妇中毫米,且没有缝合伤口应不需要。如果一个有经验的实验者做了手术。请注意这牛逼他解剖与手术有无去过夸张的在全部中的描述形象提供。这是不可能的,但有会是过度出血从的伤口如果该切割是更深比预期的,然后停止该程序和待办事项不使用的鼠标对于进一步的实验。
 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1903065--1368 Karin Westlund High 856375 \ Figs jpg \图7.jpg


7.示范图神经束入世孔处。该ç UT被夸大更好的显示在这个更高的权力观(15 X )。


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1903065--1368 Karin Westlund High 856375 \ Figs jpg \图8.jpg


图8.孔内夸张解剖(15 x )中铬肠缝合线位置的演示。


 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1903065--1368 Karin Westlund High 856375 \ Figs jpg \图9.jpg


图9.将铬色肠线与ION神经并排在圆孔中(15x )


 


用小镊子(推荐:杜蒙#5),握3 毫米缝合线通过的结束和角度它进入该洞你有无就切在该折痕中的老鼠的脸颊(图8),然后滑动的缝合进的孔,除了该。ION 的缝线是否应转到在有了非常小阻力; 如果你感觉电阻,它的手段那你待办事项不是有无的正确角度而被不指导的缝合进入的孔。该神经是否应决不是感动与该金属手术器械。
按该缝合进的卵圆作为远东作为可能与该镊子(图小号8 和9)。然后,轻轻地让围棋,而且使用的封闭技巧中你的镊子要推的结束中的缝合而且通过该孔处。你是否应就勉强要能要见的终结中的缝合如果您有无推它在正确。
该切口用于在该模型是小够了那无缝合线是必需的要关闭的伤口。在这两个沙姆斯与该FRICT-ION 模型的伤口开始要关闭在该一二十四小时(图10) 。
 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1903065--1368 Karin Westlund High 856375 \ Figs jpg \图10.jpg


图10. 深水手术在0,24,而48 ^ h 后手术。伤口是晴关闭在24 ^ h 而且是完全关闭到48 H. 只有轻微的血管分布可以被看到在24 H.  


 


放置在鼠标进入该热回收站直到它醒来从该麻醉而被移动之前返回到该家笼。老鼠应该是经过一个小时后,手术要确保所有普通汽车功能有返回。(这是一个预防措施,作为典型的小鼠是全功能在纪要中去除从麻醉。)小鼠应该然后是经过报对于该周刊继手术,其中包括评估重量和表面秋波进入的嘴要确保不加重中的伤口或者感染已经发生了。这是可能的那小鼠威尔没有增益体重的日之后的手术,但通过一天两个应该要返回到的典型的渐进重量增益。
  还有应该要没有可察觉变化在行为; FRICT-ION 小鼠应该出现类似的要朴素小鼠要的裸体,眼睛如果有是一个变化中的行为,这种由于体重下降,昏睡,等。,待办事项不使用的鼠标对于进一步的实验。如果该权重瀑布下面八十百分比中的老鼠的体重在此之前要手术,该鼠标是否应是经过由兽医人员和,如果适用,人道安乐死。


 


资料分析


 


伤害性和甲Nxiety ř 兴高采烈乙Ehavioral Ť Esting 而d 阿塔甲Nalysis


动物是否感动从该房屋间和驯化在该试验室为30 敏在他们的家笼之前要测试。动物是驯化要温和约束(持有在戴手套的手对于晶须垫测试)要评估灵敏度为机械刺激应用到的Whiskerpad ,即,反身退。经过10个培训会议要适应的啮齿动物要被举行在一个戴手套的手,灵敏度中的人脸为机械是量化到的号码中提款活动从渐变薄尼龙冯· 弗雷长丝随着定义的弯曲力(拉伸强度),刺激用的最低纤维(0.008 G,1.65)是不检测的在该后退中的人的手; 在最大的光纤(6.0 G,4.74)提供的感觉中一个钝纸剪贴线结束。动物是免费为自愿移动他们的头离开从的刺激。一个单审判下设中5个应用中冯· 弗雷长丝开始中档(0.4 G,3.61)应用一旦每3 到4 S. 如果一个积极的响应时,将下一个 较弱的灯丝是应用相反,持续直到有是没有响应; 如果没有积极的响应是诱发,在接下来更强的灯丝是应用而继续直到有是一个响应(Chaplan 的Et 铝。,1994)。该平均发生中提款活动在每个的该试验是表达作为该号码的反应出的5,0 表示没有退学,而5 指示的最大数的取款。反应要减少革兰氏力相比要控制注明提高灵敏度。经过诱导的三叉神经损伤模型,统计学显着机械过敏开发出可靠地在一个周和持续无限期作为测试用校准冯· 弗雷长丝在该ION的晶须垫感受现场(图11)。该机械刺激阈值在双方的同侧和对侧胡须垫是显着减少在小鼠后的手术相比,为了手术深水和控制小鼠。这减少持续直到安乐死,十周后 -手术。  


 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1903065--1368 Karin Westlund High 856375 \ Figs jpg \图11.jpg


图11. 雄性小鼠进行测试对于机械异常性疼痛在该晶须垫使用冯弗雷长丝。双向ANOVA (Dunnett氏多重比较试验)当时执行的在这些数据:Ñ = 5 每组,* 表示甲P -数值中< 0.05 [ 外科侧:F(20,198)= 9.178,对侧侧:F(22,144)= 32.20 ] 。在事后分析,邦费罗尼调整要所有P -值对于周逐周比较中FRICT-ION 与控制息率全部九P -值< 0.0009。


 


虽然管理中神经病理性疼痛是重要因为在许多案例这是严重的和无情的,研究为开发更好的治疗中的焦虑和抑郁共病是也中总理重要性在总体质量中人寿(冰臼的Et 铝。,2007) 。另一个主要优势的该FRICT-ION 模式是在能力要检查这些行为在慢性时间。点可测试在该模型是焦虑- 再迟来的措施这样的作为零迷宫,光暗偏好,并打开实地探索行为。在图12,从直男鼠被放置进一个浅/深商会和监控对于十几分钟。在这个测试,一个商会当时点燃有了一个明亮的灯泡虽然在其他当时不停地暗,而小鼠被允许自由访问要要么厅。小鼠随着FRICT-ION 手术是显著不太可能的要花费时间在该灯室,并探讨该商会少在通用,这是指示中焦虑。


 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1903065--1368 Karin Westlund High 856375 \ Figs jpg \图12.jpg


图12. 在模型诱导后第8周测试显示的焦虑行为(每组n = 5,*表示P 值< 0.05,使用t 检验进行比较)。


 


这两种认知与该蔗糖飞溅抑郁症测试(网络古尔13)钙ñ 是可利用的要分辩这些较高阶的后果中慢性疼痛中的FRICT-ION 型号(亚尔钦的Et 铝。,2011; Kniffin 的Et 。铝,2015年)。小鼠进行鉴于该蔗糖飞溅测试,在这30 Pasento 蔗糖水是喷涂走上老鼠的臀部,而小鼠进行监控对于五分钟要确定的号码中时报它们整齐的臀部和笼区湿润。小鼠随着FRICT-ION 手术疏导明显少常,这是指示中抑郁行为。


 


C:\ Users \ Bio-Dandan \ Dropbox \ Refomatting \ Proofreading_New提交系统\ 1903065--1368 Karin Westlund High 856375 \ Figs jpg \图13.jpg


图13. 抑郁行为进行了测试。在周8 发布模型感应在从直雄性小鼠(N = 5 每组,* 表示甲P -数值中< 0.05,比较运行使用甲Ť -Test ) 。


 


注意事项


 


对于该假手术,将小切是制造在该Bucchal 保证金而没有缝合线插入。如果完成正确,这个过程应该不诱导任何出血里面的老鼠的嘴巴还是脑腔。取出的老鼠从它的约束而将在一纸毛巾在一个干净笼子随着床上用品也已去过坐在一个暖气垫。该鼠标是否应成为自觉少比一分钟后,你有没有删除它从麻醉,而且应该要向上和散步通常在五分钟的鼠标是否应恢复从该手术有了缓和,而且要返回到正常行为通过将结束中的一天。
随着实践,这个过程是否应以没有更多比一个少数分钟。这是重要的要执行此过程快速为了避免在侧面影响中长期暴露到麻醉。
这个过程是否应被完成用无出血; 但是,如果出血时,只需应用压力到该网站有了一个棉拭子之前的出血消退。
老鼠能成为一栋在一起,而是否应不要求任何护理后手术除对标准监测。
 


致谢


 


该作者希望要承认的实验室管理技巧中凯瑟琳· 戈特,部门基金和美国国立卫生研究院资助R21 DE028096 支持的麻醉科疼痛实验室 的微创微创口内FRICT-ION 方法描述这里当时首先使用在一个最近的出版物,但我们简称为这有作为的“TIC” 法(张的Et 铝。,2019)。该FRICT-ION 协议当时改编或修改从我们以前三叉炎症压缩(TIC)方法在哪里艳色肠道缝合当时不结盟伴随的眶下神经在小鼠使用的更多微创手术方法背后的眼睛(马的Et 铝。,2012 和2015年,里昂的Et 。铝,2015年和2018)。该作者确认的洞察提供通过前研究科学家,博士妃马,谁介绍的概念中诱导眶下神经压缩通过缝合线插入进一个卵圆而是比搭售的神经要制作一个鼠标的临床前模型中慢性三叉神经性疼痛。


 


竞争利益


 


KNW 是一个ñ Unpai d 顾问为美国Elixiria 生物技术公司和一个研究生理学家在该阿尔伯克基VA 医疗保健系统。这种通信确实不必然反映的意见中的部门中退伍军人事务部或者在美国政府。


 


道德规范


 


所有的研究都设计要尽量减少动物使用而演出在按照与机构批准并在按照与该国家机构中健康指南针对的护理和使用中实验动物(NIH 出版号80-23)经修订的1996年。


 


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引用:Montera, M. A. and Westlund, K. N. (2020). Minimally Invasive Oral Surgery Induction of the FRICT-ION Chronic Neuropathic Pain Model. Bio-protocol 10(8): e3591. DOI: 10.21769/BioProtoc.3591.
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