Surgical animal models of neuropathic pain: Pros and Cons.

Just Accepted by International Journal of Neuroscience

Surgical animal models of neuropathic pain: Pros and Cons Siva Reddy Challa doi:10.3109/00207454.2014.922559

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ABSTRACT One of the biggest challenges for discovering more efficacious drugs for the control of neuropathic pain has been the diversity of the chronic pain states in humans. It is now acceptable that different mechanisms contribute to normal physiological pain, pain arising from tissue damage and pain arising from injury to the nervous system. To study pain transmission, spot novel pain targets and characterise the potential analgesic profile of new chemical entities, numerous experimental animal pain models have been developed attempting to simulate the many human pain conditions. Among the neuropathic pain models, surgical models have paramount importance in the induction of pain states. Many surgical animal models like chronic constriction injury (CCI) to sciatic nerve, partial sciatic nerve liagation (pSNL), spinal nerve ligation (SNL), spared nerve injury (SNI), brachial plexus avulsion (BPA), sciatic nerve transaction (SNT) and sciatic nerve trisection. Most of these models induce responses similar to those found in causalgia, a syndrome of sustained burning pain often seen in the distal extremity after partial peripheral nerve injury in humans. The researchers most commonly use these surgical models in both rats and mice in the drug discovery to screen new chemical entities for their efficacy in the area of neuropathic pain. However, there is scarce literature that provides comparative discussion of all these surgical models. Each surgical model has its own benefits and limitations. It is very difficult for a researcher to choose a suitable surgical animal model to suit their experimental set-up. Therefore, particular attention has been given in this review to comparatively provide the pros and cons of each model of surgically induced neuropathic pain.

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Surgical models of neuropathic pain in small animals

Siva Reddy Challa*

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Surgical animal models of neuropathic pain: Pros and Cons

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Vijayawada-520 010, India

*Corresponding author

Professor & HOD,

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Department of Pharmacology

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Siva Reddy Challa, M.Pharm, Ph.D

KVSR Siddhartha College of Pharmaceutical Sciences, Pinnamaneni Poly Clinic Road, Siddhartha Nagar

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Vijayawada-520010 Andhra Pradesh , India.

Mobile No: +918008593009

Email: [email protected]

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Department of Pharmacology, KVSR Siddhartha College of Pharmaceutical Sciences,

ABSTRACT

One of the biggest challenges for discovering more efficacious drugs for the control of neuropathic pain has been the diversity of the chronic pain states in humans. It is now acceptable that different mechanisms contribute to normal physiological pain, pain arising from tissue


damage and pain arising from injury to the nervous system. To study pain transmission, spot novel pain targets and characterise the potential analgesic profile of new chemical entities,

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numerous experimental animal pain models have been developed attempting to simulate the many human pain conditions. Among the neuropathic pain models, surgical models have paramount importance in the induction of pain states. Many surgical animal models like chronic

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ligation (SNL), spared nerve injury (SNI), brachial plexus avulsion (BPA), sciatic nerve transaction (SNT) and sciatic nerve trisection. Most of these models induce responses similar to those found in causalgia, a syndrome of sustained burning pain often seen in the distal extremity

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after partial peripheral nerve injury in humans. The researchers most commonly use these

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surgical models in both rats and mice in the drug discovery to screen new chemical entities for their efficacy in the area of neuropathic pain. However, there is scarce literature that provides comparative discussion of all these surgical models. Each surgical model has its own benefits and limitations. It is very difficult for a researcher to choose a suitable surgical animal model to

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suit their experimental set-up. Therefore, particular attention has been given in this review to comparatively provide the pros and cons of each model of surgically induced neuropathic pain.

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constriction injury (CCI) to sciatic nerve, partial sciatic nerve liagation (pSNL), spinal nerve

INTRODUCTION An animal model is well utilized for research because it has specific features that resemble

a human disease or disorder. These features can either be spontaneous or induced. Though the understanding about the fundamental mechanisms for neuropathic pain in humans is still incomplete, a number of animal models of peripheral nerve injury might imitate human neuropathic pain signs, e.g. hyperalgesia, allodynia and spontaneous pain, making these animal


models important tools for neuropathic pain experiments [1]. The existing animal models are distinguished by both location and form of injury. Numerous animal models for nerve damage

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and the subsequent neuropathic pain have been developed over the last decades. Here, focus is on the differentiation of all these surgical models with their advantages and disadvantages. However, this review is mainly limited to spinal nerve injury models and other surgical models

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Surgical models of neuropathic pain Chronic constriction injury (CCI) to sciatic nerve:

Chronic constriction injury simulates the symptoms of chronic nerve compression in clinical

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conditions of trauma or tumor developments. This model also mimics a lesion of nerve fibres

tunnel syndrome).

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located mainly at the surface of the peripheral nerve (a typical feature of, for example, carpal

Procedure: In CCI model, a 1.5-cm incision is made 0.5 cm below the pelvis. The biceps femoris and the gluteous superficialis are separated and the sciatic nerve is exposed and isolated

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and four loose ligatures (5-0 chromic catgut) with 1-mm spacing are placed around it. CCI also results in the development of allodynia like behavior, hyperalgesia and spontaneous pain like behaviors, which normally reach maximum 10-14 days after surgery [1, 2,3,4]. In this model,

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of trigeminal neuralgia are not covered in this review.

chromic catgut contributes to the inflammatory component in addition to neuropathic pain [5]. Merits:

1. It is reliable and easily reproducible model 2. It is a frequently used model particularly for behavioral studies. 3. It results in intraneural oedema, which strangulates the nerve, effectively axotomizing many but not all of the nerve axons


4. It mimics the post traumatic peripheral painful neuropathy states in humans. 5. Partial denervation preserves some behavioral responses to peripheral stimuli.

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6. It shows behavioral signs of spontaneous pain. 7. This model produces a greater reduction (almost all Aβ fibres) and very large majority of Aδ fibres are axiotomized while large numbers of C-fibres are intact.

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9. This is suitable model for assessing cold allodynia when compared to partial sciatic nerve ligation (PNL) and spinal nerve ligation (SNL).

10. This model involves both neuropathic and inflammatory components

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Demerits:

the study.

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1. Autotomy leads to damage of digits of hind paws and renders the animal not suitable for

2. The tension of the ligatures around the sciatic nerve can vary and this will obviously influence the number and type of injured afferent fibres.

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3. Variability of degree of damage from animal to animal due to variation in the snugness of ligations even by the same experimenter in different animals. 4. It is clear that the degree of allodynia produced in this model in less when compared to

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8. This model involves both inflammatory component and neuropathic component.

other models like partial sciatic nerve ligation and spinal nerve ligation probably due to the more damage to Aβ fibres (which are mainly responsible for allodynia) causes disappearance of allodynia. Intact C fibres may account for the mechanical and thermal hyperalgesia.

5. This model exhibits thermal hyperalgesia (which is not a symptom of clinical neuropathic pain).


Spinal nerve ligation (SNL) to sciatic nerve: Procedure:

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An incision is made along the spinal column and the left paraspinal muscles are separated from the spinous processes at the L4-S2 levels. Under a modular high-performance stereomicroscope, the L5 spinal nerve is isolated and 1–3mm of the nerve is ligated distal to the

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Merits:

1. Partial denervation preserves some behavioral responses to peripheral stimuli 2. This model is suitable for the selective study of injured and uninjured nerve fibres in the

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sciatic nerve. Here, L5 & L6 account for the injured axons of the sciatic nerve while L-4

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constitutes for uninjured axons of sciatic nerve.

3. The effective damage is equal in afferent axons of all sizes. SNL surgical procedure tightly ligates the same spinal nerves in each animal. 4. Experimental variability is less since the same number of nerve fibres will be damaged in

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each animal unless there is biological variability from animal to animal in terms of contribution of three spinal nerves in the formation of sciatic nerve. 5. Excellent model for the assessment of mechanical allodynia and it also shows symptoms

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dorsal root ganglia. Special care should be taken to avoid any damage to the L4 spinal nerve [6].

of hyperalgesia to less extent.

6. Autonomy is absent in this model. 7. It is also an excellent model for in-vitro use as the damaged and undamaged fibres of the peripheral nerve originate from distinct DRGs

Demerits:


1. Surgical procedure of this model is complex, time consuming and needs experimental skill to avoid the damage to L-4 nerve while ligating L-5 & L-6. Even slight damage to

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the L-4 nerve abolishes the responses of allodynia. In addition, it causes severe motor deficit and interferes with behavioral tests since it has an abundance of motor fibers. 2. In this model, fairly extensive surgery with muscle damage might complicate the

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Partial sciatic nerve ligation (pSNL): Procedure:

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The experimental procedure involves the ligation of the ipsilateral sciatic nerve at the highthigh level, so that 1/3–1/2 thickness of the sciatic nerve is trapped in the ligature. PNL rats

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exhibit signs of allodynia to von Frey hair stimulation and hyperalgesia to both thermal and mechno-noxious stimuli within hours of ligation; the symptoms last for over 7 months. Ligated rats also display signs of spontaneous pain in the forms of paw guarding and licking on the injury

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side. The evoked pain can develop into bilateral patterns [7]. This partial nerve ligature model simulates a nerve contusion rather than nerve compression. Merits:

1. Partial denervation preserves some behavioral responses to peripheral stimuli.

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pathomechanism.

2. Damage is equal to axons of all sizes. 3. It is a model of moderate symptoms of mechanical allodynia, cold allodynia and symptoms of hyperalgesia. 4. It is an easy surgical procedure and consumes less time when compared to SNL model.


5. High reproducibility and ease of surgical procedure are two important advantages of this model.

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Demerits: 1. Autonomy leads to damage of digits of hind paws and renders the animal not suitable for the study.

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the same experimenter cannot maintain the consistency of ligature of equal number of nerve fibers every time.

3. It is difficult to study changes in the Dorsal Root Ganglion (DRG) as damaged and

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Spared Nerve Injury (SNI):

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undamaged primary afferents are mixed in the nerve.

Surgical Procedure: An incision is made at the lateral surface of the left thigh, and the proximal and distal parts of the biceps femoris muscle are separated to expose the sciatic nerve and its three terminal branches. The tibial and common peroneal nerves are tightly ligated and 2–3 mm

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of the nerves distal to the ligation removed. Any stretching or contact with the spared sural nerve should be avoided. The model affords high reproducibility. It is easy and fast to perform with a high operation success rate even for inexperienced operators. The SNI operated animals have

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2. There is lot of variability in ligating the number of nerve fibers per each animal and even

normal food intake, movements, grooming and growth [8].

Following SNI surgery, both rats and mice as early as two to three days after injury will

develop long-term hypersensitivity to mechanical, but not to thermal stimuli [9, 10]. For rats, the hypersensitivity is of long duration (15 months or longer) [11]. For mice, the duration is at least 30 days [10]. An increased sensitivity to cold stimuli has also been observed in SNI-operated


rats. The SNI model differs from the SNL model in that the intermingling of intact and injured neurons in peripheral target tissues is restricted to the border territory between the lesioned tibial

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nerve and intact sural nerve. Merit:

1. This permits behavioral testing of the non-injured sural nerve territory (adjacent to the

following injury of neighboring nerves. Demerit:

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take place in the peripheral afferent terminals of the intact sural afferent neurons

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1. This model induces lesions in the peroneal and the tibial nerves leaving the sural nerve

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intact are that the procedure causes a degeneration of axons, which also limits the number of distal intact axons. Hence it will lead to difficulties in the performance of behavioral tests, as the ipsilateral paw includes uninjured areas close to the denervated areas. 2. An additional complication for the assessment of pain behavior in SNI animals is that the

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hypersensitive area in the limb is the lateral part of the paw that is technically difficult to test.

Brachial plexus avulsion (BPA) model:

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denervated areas) and also enables to investigate the neuro- physiological changes which

BPA model mimics a frequent type of human nerve traction injury following traffic

accidents.

Surgical Procedure: This surgical procedure is usually performed following anesthesia induced by a 7% chloral hydrate solution (0.6 ml/100 g body weight) injected intraperitoneally. The brachial plexus is


approached through a horizontal incision parallel to the clavicle, running from the sternum to the axillary region. The pectoralis major muscle is displaced, leaving the cephalic vein intact. The

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subclavian vessels are located and the lower trunk of the brachial plexus is isolated from surrounding tissues. In the avulsion group of rats, the lower trunk is grasped with forceps and extracted from the spinal cord by traction. In the sham operated group, the brachial plexus is

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and the skin closed with 4–0 silk sutures. It has been reported that in humans, the brachial plexus avulsion (BPA) induces a constant crushing and intermittent shooting pain that remains without an adequate treatment [14]. This lesion may lead to pathological plasticity of the CNS that is

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associated with altered pain sensations [15]. The main characteristics of BPA are the rapid onset

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of pain (an effect which occurs immediately after the trauma) and the long-lasting development of neuropathy, which may be evidenced distant from the site of the lesion, either on the ipsilateral or contralateral side [16, 12].

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Merits:

1. It is a new and reliable model for the study of neuropathic pain 2. In this model, the neuropathy can be detected even at distant sites from the injury, both in ipsilateral and contra-lateral hind paws.

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exposed without any lesion to the nerves [12, 13]. The cut tissue layers are then approximated

3. The avulsion of brachial plexus in mice was not associated to autonomy. 4. It is a valid model for studying the long lasting mechanical allodynia and cold allodynia that typifies humans with a similar injury.

Demerit: 1. This model does not produce thermal hyperalgesia


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Sciatic Nerve Transection model This is the oldest model of neuropathic pain in animals and involves complete transection of sciatic nerve at mid-thigh level [17]. The sciatic nerve transection model simulates the clinical

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spinal lesions. Surgical Procedure

In this model, rat is anesthetized and the common sciatic nerve is exposed. The

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connective tissue attached to the sciatic nerve is cleared off and the sciatic nerve is firmly tied by

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nylon suture, proximal to its bifurcation into the tibial and the peroneal divisions, at two locations about 1cm apart. The nerve is then completely transected between the pair of ligatures and 5 mm of the nerve between the ligatures is removed to prevent the re-joining of nerves due

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to regeneration. The adjacent sapehnous nerve is also lesioned to induce entire denervation of distal hind limb. Following complete nerve transection, a neuroma develops at the proximal nerve stump consisting of regenerative nerves sprouting in all directions [18, 19, 20]. The model produces anesthesia dolorosa, i.e. pain in the area in the absence of any sensory input in that

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symptoms of “phantom limb”, a condition that arises in humans after amputation or transversal

area. Autotomy (self-attack and mutilation of the denervated limb by injured animals) is observed in this model and often considered as a marker of neuropathic pain [21]. The degree of autotomy in-turns depends upon the method and location of neurectomy [22]. There has been considerable debate whether autotomy is a reflection of spontaneous pain or is a result of excessive grooming in the absence of sensory feedback [23].


Merits:

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1. It is suitable model for simulating phantom limb pain 2. The model produces anesthesia dolorosa, i.e. pain in the area in the absence of any sensory input in that area [22].

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1. Ethical considerations are also the key issues in this model as animals demonstrate excessive autotomy in this model ( [24].

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Spinal Nerve Transection (SNT) model Procedure

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An incision is made along the spinal column and the left paraspinal muscles are separated from the spinal processes at the L4-S2 levels. Under a modular high-performance stereomicroscope, the L5 spinal nerve is isolated and 1–3mm of the nerve is excised distal to the

Merit:

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dorsal root ganglia [25].

1. It is a old model and may simulate the phantom limb pain in humans Demerit:

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Demerit:

1. This model lacks the local inflammatory component that is present in the CCI, partial sciatic nerve ligation (PSNL) and SNL models

2. Assessment of pain behavior is difficult in this model.

CONCLUSION


All these surgical models differ in several important respects. For example, the PSL and SNL models decrease the number of sciatic afferents that innervate the periphery by about one-

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half, with afferents of all sizes affected about equally. The CCI model induces a larger reduction that is dissimilar with respect to size (nearly all the A-beta fibers and a very large majority of the A-delta fibers are axotomized, while large numbers of C-fibers are intact). Therefore, the

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activation of A-beta low threshold mechanoreceptive afferents, while the mechano-allodynia of the CCI model must be due to activation of A-delta and/or C fibers. Besides, the location of the axotomy relative to the afferent’s cell body in the dorsal root ganglion is very different quite

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close in the SNL model, but quite distant in the pSNL and CCI models. An obvious neuroma-

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incontinuity develops in the PSL, and this is also the only model where the epineurium is deliberately breached (important because of the known differences between nerve transection and nerve crush). Which is the best model? There is no “best” model. Human painful peripheral neuropathies are very diverse. No single animal model will answer all questions about the human

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conditions. but all of these neuropathic pain models have contributed significantly to our study of neuropathic pain.

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mechano-allodynia seen in the PSL and SNL models is expected to be at least partly due to

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