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Industrial Sensors for Hundreds of Applications. The smooth transition between stimulation levels allows you to gradually increase stimulation and find the exact level that your dog needs.

To recondition your batteries you should charge the system completely as stated in your Owner's Manual typically 14 continuous hours for non-G2. In about 10 days time after i sent them in i received what appeared to me two new collars, transmitter, beepers, etc.

Product Specifications Product specifications may change without notice or obligation since Tri-Tronics is committed to a policy of continuous improvement. The Garmin Pro is a new and improved version of the Tri-Tronics Pro G3, which have been a favorite product of both professionals and every day users, who have relied upon the these products for many years. It still contains all of the quick change features trainers have always appreciated in the Pro However, with the use of animal models come challenges relating to the appropriate quantification of behavioral responses that could be considered equivalent to pain in humans.

Despite some degree of uncertainty about the validity of the anthropomorphization of pain in animals, the capacity to experience pain and distress, particularly resulting from procedures or conditions that would cause pain and distress in humans, must be assumed unless there is evidence to the contrary.

Undoubtedly, nociception, or the ability to detect a potentially harmful stimulus, is a fundamental physiological function in mammals and indeed many other species.

It should be noted that no test can therefore measure pain in animals directly—the presumably unpleasant emotional experience of pain is inferred from pain-like behaviors which can include the withdrawal of a body part from a stimulus, reduced ambulation, agitation, an increase in grooming of the affected area, and vocalizations upon sensory stimulation. The distinction between nociception and pain thus underlines a key difference in terminology when referring to communicating and non-communicating subjects.

Similarly, as it cannot be said that the animal feels pain, analgesia and analgesic intervention cannot take place—only anti-nociception and anti-nociceptive interventions can. Accordingly, as pain cannot be directly measured in rodents, it has been necessary to develop indirect methods to quantify and evaluate pain-like behaviors in non-anesthetized animals which are reliable, reproducible, sensitive and specific Mogil, This review article will provide an overview of the current behavioral methods that are used to assess pain behaviors in mice and rats.

The term nociception was coined by Charles Sherrington in the early s to distinguish the sensation of pain—a result of central nervous system processing—from the physiological phenomenon of the peripheral nervous system responding to potential harmful stimuli Dubner, ; Coutaux et al.

Thus, the term nociception is used to describe the peripheral neuronal response to noxious stimuli, which encompasses any stimuli, being mechanical, thermal, electrical or chemical, that have the potential to damage are damaging to tissue Dubin and Patapoutian, Typically, noxious stimuli activate nociceptors, a subset of peripheral sensory neurons, which have a range of specialized ion channels and receptors that transduce noxious stimuli into electrical signals.

These neurons are pseudo-unipolar, with a peripheral branch that terminates in the skin or viscera and a central branch that terminates in the spinal cord. Nociceptive signals are then sent to the spinal cord and brain for processing as the sensation of pain. Thus, pain is an experience that encompasses both sensory and emotional components; therefore the term pain is not interchangeable with nociception. In human patients, a distinction is made between stimulus-evoked pain and stimulus-independent or spontaneous pain.

Stimulus-evoked pain is described as either hyperalgesia or allodynia, and is further subdivided on the basis of the evoked stimulus modality e. Hyperalgesia is defined as an increased or exaggerated pain response to a normally noxious stimulus, while allodynia is defined as a painful response to a normally non-noxious or innocuous stimulus.

In cases of sensory loss, hypoalgesia may be present, which is defined as decreased sensitivity to a nociceptive stimulus. Stimulus-evoked pain can be evaluated in humans using quantitative sensory testing.

While not in routine clinical use, quantitative sensory testing has the potential to improve patient outcomes by classifying pain based on the mechanism and choosing treatments that target that mechanism Baron et al. Stimulus-independent or spontaneous pain may be paroxysmal sudden and severe or continuous, and can be described as aching, cramping, crushing, shooting and burning Jensen et al. Importantly, the pain appears to be spontaneous, with no identifiable stimulus. However the distinction between stimulus-evoked and non-stimulus evoked pain may be difficult to make clinically, as arguable it could be allodynia occurring from an unidentified stimulus.

Mechanical hyperalgesia and allodynia can be further subdivided into dynamic triggered by brushing , punctate triggered by touch and static triggered by pressure. Dynamic mechanical allodynia and hyperalgesia can be assessed by brushing the skin with a cotton bud, paintbrush or cotton ball, and in the case of allodynia, can be evoked by the brushing of clothing, bed sheets or towels against the skin Jensen and Finnerup, Punctate mechanical allodynia and hyperalgesia can be evoked with a pinprick or monofilament, and in practice can be assessed by the application of von Frey filaments of varying forces 0.

Static hyperalgesia can be superficial or deep and is assessed by the application of pressure to the skin or underlying tissue by a finger or using a pressure algometer Jensen and Finnerup, The exposure of peripheral sensory nerve endings to elevated temperatures can evoke sensations of warm, hot, or pain.

As for heat pain, these values are likely influenced by a number of factors including ambient temperature, rate of cooling and anatomical location. Given the variability in cold pain thresholds, it may be difficult to differentiate between cold allodynia and cold hyperalgesia in the clinic. International standards and guidelines, as well as country-specific codes and legislation, have been developed to protect the welfare of animals used for research. In fact, it is a requirement for publication of in vivo data in high quality journals that relevant standards and guidelines are strictly adhered to McGrath et al.

The framework for these standards and guidelines are based on the principles of the 3Rs replacement, reduction, refinement. According to the replacement principle, the use of live animals should be replaced with in vitro or computational methods where possible, and if unavoidable, the use of non-sentient or less sentient animals is preferred.

However, replacement or substitution of animals for nonsentient materials is difficult in pain research due to the nature of the behavioral experiments. Thus, the focus is often on reduction of the number of animals necessary to obtain data, and refinement of the method with the aim to decrease the amount of nociception caused to the animal.

This can be achieved through a variety of techniques. For instance, improving data homogeneity and enhancing statistical power Dell et al.

Similarly, measures to improve data quality, including appropriate randomization and blinding procedures, are key to ensure validity of the obtained results. In addition, care should be taken to design experiments that minimize distress and suffering.

This includes minimizing the duration of models, replacing nocifensive model compounds for ones that cause shorter lasting nociception, or reducing the administered doses of compounds.

Particular mention should also go to timely publication of data, be it positive or negative results, in order to reduce experimental duplication and unnecessary use of animals. If a stimulus is applied that does not normally evoke a withdrawal response, and the animal withdraws from the stimulus, the animal is considered to have allodynia.

Similarly, if a stimulus is applied that does normally evoke a withdrawal response, but the animal withdraws with an exaggerated response, the animal is considered to have hyperalgesia.

However, in practice it is difficult to distinguish between allodynia and hyperalgesia in animals, and the terms allodynia and hyperalgesia are often used incorrectly or interchangeably in the literature.

Similarly, the terms nociception and pain are often used interchangeably, although the term pain is rarely appropriate to use in reference to animal studies. The outcomes of most behavioral methods used to study nociception are somewhat subjective.

For example, in the case of application of a stimulus to the hind paw, the investigator must determine if the animal withdrew the hind paw due to its aversive nature, or whether the animal withdrew the hind paw for another reason e. Behaviors tend to occur on a spectrum of intensity, but are usually scored in binary as either present or absent. As each researcher cultivates a slightly different cut off point in their minds as to what constitutes a behavior on this spectrum, results can vary significantly between laboratories.

Similarly, human scoring lends itself to bias, although this can be avoided with appropriate randomization, allocation concealment and blind outcome assessment Hirst et al. It should be noted that behavioral assessment of animals in groups even if blinded is typically not sufficient, with a preferred method being measurements performed on animals in random order by an investigator blinded to the treatment group each animal has been allocated to.

The behavioral methods used to measure nociception in rodents can be divided into stimulus-evoked and further subdivided by the stimulus modality—mechanical, heat, cold and non-stimulus evoked, with the most commonly used methods discussed in this review article. For detailed protocols of these methods see Minett et al. For an overview of commonly used pain models in rodents see Gregory et al. The presence and extent of aversive behaviors in responses to mechanical stimuli is typically determined using manual or electronic Von Frey or the Randall Selitto test, as described below Figures 1A—C.

Figure 1. Methods used to assess mechanically evoked pain like behaviors in rodents. A Manual Von Frey. Rodents are placed individually in small cages with a mesh or barred floor. Monofilaments of differing forces are applied perpendicularly to the hind paw.

If the rodent withdraws, licks or shakes the paw, it is considered to have had a positive response. Rodents are placed individually in a small cage with a barred floor. A single, un-bending filament is applied perpendicularly to the hind paw. The force is increased by rotation of the handheld device until paw withdrawal occurs. The force ramp and paw withdrawal force are displayed by the software post-test. C Randall-Selitto test handheld device.

The rodent is restrained and the hind paw or tail is placed between a pointed probe tip and flat surface. The pressure is increased until withdrawal or vocalization occurs. The manual Von Frey test, developed by the physiologist Maximilian von Frey, is a method of evaluating mechanical allodynia in mice and rats.

Despite the development of electronic Von Frey tests, manual Von Frey remains the gold standard for determining mechanical thresholds in mice. In this test, animals are placed individually in small cages with a mesh or otherwise penetrable bottom. A monofilament is applied perpendicularly to the plantar surface of the hind paw until it buckles, delivering a constant pre-determined force typically 0. A response is considered positive if the animal exhibits any nocifensive behaviors, including brisk paw withdrawal, licking, or shaking of the paw, either during application of the stimulus or immediately after the filament is removed.

While the plantar surface of the hind paw is the most commonly used area for testing, other areas of the body, including the dorsal surface of the hind paw or the abdomen can also be used Minett et al. If there is no response, the next filament with a higher force is tested; if there is a response, the next lower force filament is tested.

A disadvantage of this method is that the number of measurements per animal is variable and that it requires repeated, time-intensive measurements, which may lead to sensitization or learnt responses.

Figure 2. Visual representation of the different methodological approaches used to determine mechanical sensitivity using manual Von Frey. This continues until at least four readings are obtained after the first change of direction. The test begins by assessing the response to a filament of the lowest force in this case 0. In this test, monofilaments of varying forces in this case 0. It is an estimate, as the force applied by manual von Frey filaments can only be applied in discrete steps, and not continuously like electronic von Frey see below.

The method is based on the application of monofilaments with increasing force until a withdrawal response is elicited, and the force of the von Frey filament that elicits this positive response is designated as the mechanical withdrawal threshold Figure 2B. An advantage of this method is that it avoids excessive application of Von Frey filaments that elicit aversive behaviors. The advantage of this approach is that each animal receives the same number and type of stimuli, although the number of tests per hind paw could exceed 50 e.

The manual Von Frey tests enable quantification of mechanical thresholds in unrestrained animals, which removes the risk of handling-induced stress.

However, this also requires animals to be acclimatized to the cages, to ensure ambulation and exploratory behaviors, which could be misinterpreted as a positive response, are kept to a minimum. Testing should also be avoided while the animal is engaged in grooming behaviors, as this can produce false negative responses.

Consistent and precise placement of the filament is important to reduce intra-subject and inter-subject variability, with the specific placement dependent on the innervation territories of the test area and the model used.

For example, in the spared nerve injury model, the tibial and common peroneal nerves are axotomized, leaving only the sural nerve intact. In this model, the lateral plantar skin of the hind paw, which is the area of innervation of the sural nerve, has the greatest reduction in mechanical thresholds compared to other innervation areas of the plantar skin Decosterd and Woolf, A touch-on reaction is more likely to occur if the filament is not applied perpendicularly, if the filament is not applied smoothly, or if the filament moves horizontally during application, inducing scratching.

In addition, rodents are intelligent and can learn that premature withdrawal will result in less human interaction and stimulation. Electronic Von Frey systems operate under similar principles as manual von Frey, except that a single, un-bending filament is applied with increasing force until a paw withdrawal response is elicited. The force at which this response occurs is recorded automatically by the apparatus and is designated as the paw withdrawal threshold. The main advantage of electronic Von Frey compared to manual Von Frey is that an increasing force is applied by a single filament.

This therefore provides measure of paw withdrawal threshold on a continual scale, as the force is applied continuously and not in steps. In addition, the experimental time is dramatically reduced, as few applications usually 3—4 are needed to determine the paw withdrawal threshold Deuis et al. In addition, as for manual Von Frey tests, animals still need to be habituated to the cages until exploratory behaviors have ceased. The Dynamic Plantar Aesthesiometer or Plantar Von Frey , houses rodents in an enclosure with a mesh screen floor, under which a movable touch-stimulator unit is placed.

Under the direction of the researcher, the apparatus applies a von Frey 0. The device automatically records the force at which paw withdrawal occurs and the rate at which the force is applied can be changed. In contrast to other Von Frey setups, animals are housed in individual enclosures with bars, rather than mesh, to help maximize the surface area of the hind paw available for application of the filament.

However, as the testing surface can influence the results of von Frey, it is possible that values obtained using MouseMet or RatMet are not directly comparable to other methods Pitcher et al. In addition to displaying force at which paw withdrawal occurs, the rate at which the force was applied is also displayed post-test by the software, to ensure the force ramp was applied consistently.

Both instruments have been validated against manual von Frey filaments and found to produce less variable data in addition to being easier to use. It should be noted that the absolute values obtained using manual and electronic Von Frey can differ significantly. While the underlying protocols and principles are different, it remains to be determined if electronic Von Frey activates a different subset of sensory neurons compared to manual Von Frey e.

However, irrespective of the method used, the endpoint is paw withdrawal to a stimulus that is not normally aversive, and thus both methods can measure mechanical allodynia. The Randall-Selitto or paw pressure test was developed as a tool to assess response thresholds to mechanical pressure stimulation and is often considered a measure of mechanical hyperalgesia Figure 1C ; Randall and Selitto, This test involved application of an increasing mechanical force to the surface of the paw or tail until withdrawal or vocalization occurs.

In practice, this test is useful for assessment of nociceptive thresholds in rats rather than mice as animals need to be heavily physically restrained with the tested paw held out, and mice rarely tolerate such handling Anseloni et al.

The exception is use of the test on the tail of mice Minett et al. The Randall-Selitto test can be performed using bench-top e.

To obtain reliable data, animals need to be habituated to the restraint method and experimental apparatus, which can become very time-intensive. Mechanical pressure is applied focally to the dorsal or plantar surface of the hind paw or tail, which is placed between a pointed probe tip and a flat surface.

The pressure is then increased at a constant rate until a nociceptive behavioral response is observed. The withdrawal response is detected visually by the researcher, resulting in subjective measurement of the threshold. It should be noted that the paw withdrawal threshold can be considered a measure of spinal reflex, with some researchers favoring vocalization as an end-point Winter and Flataker, ; Kayser and Christensen, ; Santos-Nogueira et al. These measures can have a profound effect on the apparent anti-nociceptive efficacy of test compounds and should thus be carefully considered during experimental design Winter and Flataker, However, rodents do not vocalize in the audible range unless the pain is severe, making use vocalization as an endpoint ethically limited Mogil, The use of ultrasonic inaudible vocalization as an endpoint has also been studied, however it has not consistently been shown to increase in response to noxious stimuli Han et al.

While the Randall-Selitto test often results in similar types of outcomes to the Von Frey filament tests Santos-Nogueira et al. Both versions of the test require the animal to be loosely restrained. Figure 3. Methods used to assess heat-evoked pain like behaviors in rodents. A Tail flick test radiant heat. Rodents are restrained and a focused beam of light is applied to tail. B Hot plate test. C Hargreaves test. Rodents are placed individually in small enclosures with a glass floor.

A radiant or infrared heat source is focused on the plantar surface of the hind paw and the time taken to withdraw from the heat stimulus is recorded.

D Thermal probe test. Mice are placed individually in small cages with a barred floor. A small metal probe is applied to the hind paw, and heating is triggered by rotation of the handheld device until the mouse withdraws the paw. While relatively quick and easy to perform, an important consideration with the tail flick test is that a similar behavioral response can be observed in spinally transected rats, consistent with the notion that the tail withdrawal response is a spinal reflex, rather than an indication of pain behaviors involving higher brain centers Irwin et al.

This suggests that the tail flick response may be impacted by changes in motor processing Chapman et al. In addition, skin and ambient temperature, the location of stimulus application on the tail, as well as learnt avoidance behaviors can affect the withdrawal response Yoburn et al. The clinically translatability of the tail flick test is therefore unclear. While the method carries the disadvantage that the rodent has to be restrained, the tail flick test is of very short duration so handling can be minimized easily.

The hot plate test, first described in , can be used to determine heat thresholds in mice and rats Woolfe and Macdonald, Unlike the tail flick test, the hot plate test and other tests that apply heat stimuli to the hind paws are considered to integrate supraspinal pathways, as rats with spinal transection do not withdraw the hind limbs in the hot plate test Giglio et al.

Nocifensive behaviors include forepaw withdrawal or licking, hind paw withdrawal or licking, stamping, leaning posture and jumping Espejo and Mir, While forepaw withdrawal often occurs first, hind paw withdrawal or licking is considered to be a more reliable indicator of nociception, as the forepaws are frequently used in grooming and exploration and are not consistently in contact with the metal surface Woolfe and Macdonald, ; Minett et al.

If no nocifensive behaviors are observed, the animal must be removed from the hot plate after pre-determined cut-off time to prevent tissue damage.

Alternatively, the number of flinches over a set period of time can be recorded at a specific temperature Yalcin et al. The dynamic hot plate test, first described in , uses an increasing temperature ramp rather than a constant temperature. The temperature at which this occurs is designated as the response temperature Ogren and Berge, ; Tjolsen et al. The response temperature is dependent on the starting temperature, ambient temperature and rate of heating, with faster heat ramps resulting in higher response temperatures Tjolsen et al.

As for the static hot plate test, cut off times should be carefully designed and strictly adhered to in order to avoid unnecessary nociceptive stimulation and tissue damage.

Depending on the species and strain of rodent used, at least 12 different behaviors have been noted in the hot plate test, including sniffing, grooming, stamping of the legs, freezing, licking, leaning and jumping Espejo and Mir, Some of these behaviors can be sensitive to analgesics, although differences are observed depending on the type of behavior quantified.

For example, paw licking is diminished by opioids but not other analgesics, while other behaviors can also be affected by other classes of analgesics Ankier, ; Hunskaar et al.

Data quality is usually improved if the time to occurrence of any behavior, rather than specific behavior types, is recorded, and if lower temperatures are used Carter, ; Plone et al. It is plausible that differences in behavior may relate to the type of sensory fiber activated. An additional confounding factor in the hot plate test is the tendency for learned behavioral responses, which lead to diminished reaction times during subsequent exposures to the hot plate Gamble and Milne, ; Plone et al.

Thus, the hot plate test can produce greatly variable data, even within laboratories. An additional disadvantage of the hot plate test is that all four paws and the tail are exposed to the heat stimulus. While this is generally not an issue when testing the anti-nociceptive effects of compounds delivered systematically or when phenotyping transgenic mice, this may confound the results for unilateral models of pain or for compounds administered by intraplantar injection.

The Hargreaves test, first described in , is a method used to quantify heat thresholds in the hind paws of mice and rats upon application of a radiant or infrared heat stimulus Hargreaves et al. The Hargreaves test is usually carried out using a glass bottom enclosure, which can be heated to minimize errors arising from heat sink effects.

A radiant or infrared heat source is positioned underneath the animal and aimed at the plantar surface of the hind paw Figure 3C. The time taken to withdraw from the heat stimulus is recorded as the withdrawal latency, and depending on the model of the Hargreaves apparatus, may either be recorded manually by the investigator or automatically by the apparatus.

The Hargreaves test permits measurement of ipsilateral and contralateral heat thresholds, allowing each animal to serve as its own internal control in unilateral pain models. In addition, the Hargreaves test enables quantification of heat thresholds in unrestrained animals, reducing the likelihood of stress-induced responses. However, this requires the animals to be acclimatized to the apparatus to minimize ambulation so that withdrawal latencies can be accurately determined.

While generally not an issue in rats, which are reported to only require 5 min of habituation Hargreaves et al. A disadvantage of the Hargreaves test is that the paw withdrawal time is recorded rather than directly measuring the paw withdrawal temperature. While paw withdrawal temperature can be derived from the time to withdrawal Hargreaves et al. To address this, a modified Hargreaves test has been reported, that utilizes a feedback-controlled radiant heat source to apply a constant temperature to the hind paw.

While validated using an incisional model of pain in rats, this method takes longer to perform than the normal Hargreaves test and is not available to purchase commercially. The thermal probe test MouseMet Thermal, Topcat Metrology is a novel method recently described to quantify heat thresholds in mice Deuis and Vetter, This test can be carried out using the same mouse enclosures as the electronic von Frey test MouseMet and is based on the application of a 2 mm thermal probe to the hind paw.

The temperature at which paw withdrawal occurred is automatically recorded, enabling recording of the paw withdrawal temperature without the delay of an investigator manually noting the temperature Deuis and Vetter, Similar to the Hargreaves test, the thermal probe tests enables quantification of ipsilateral and contralateral heat thresholds in unrestrained mice, but with a shorter habituation time of 5—10 min.

The main advantage of the thermal probe test is that the mice are placed in individual runs standing on bars instead of glass enabling access to the plantar surface, which allows simultaneous assessment of mechanical thresholds by von Frey, removing the need for acclimation in two different enclosures.

While application of a contact heat stimulus achieves consistent and efficient thermal transfer, it also represents a mechanical stimulus that may lead to premature paw withdrawal in models with mechanical allodynia. Accordingly, the development of thermal allodynia was demonstrated to be independent to the development of mechanical allodynia in unilateral models of carrageen-induced inflammation and burn injury Deuis and Vetter, Nonetheless, the thermal probe test remains to be validated in other pain models that cause more pronounced mechanical allodynia.

A major advantage of the thermal probe test is the reduced time required for acclimatization to the testing environment, enabling characterization of models or compounds with short duration of action, as well as testing of mechanical and thermal thresholds in the same enclosure. In addition, welfare benefits in form of testing of unrestrained mice and exposure of only a single hind paw to a noxious heat stimulus are favorable. A modified version of the thermal probe test suitable for quantifying heat thresholds in rats is still to be developed.

The cold plate test is one of the simplest assays to determine behavioral responses to both noxious and innocuous cold temperatures in both mice and rats. A number of endpoints can be obtained from the cold plate test, similar to the hot plate test. Here, the rodent is placed on the plate after it has been cooled to the desired temperature and the time taken to evoke nociceptive behavior such as shaking, jumping or licking in the animal is recorded as the response time.

Second, the number of flinches over a set period of time can be recorded at a specific temperature Deuis et al. Third, aversive response to a cooling ramp can be used to determine the cold response threshold Yalcin et al. It should be noted that rather than flinching or licking, some rat strains tend to simply avoid weight bearing on the affected paw or reposition their stance to minimize contact with the cool surface, so all observation should be adapted to the specific model animal.

These techniques provide an insight into how sensitive to cold temperatures the animal is, and thus provides an indirect measure of cold-induced hyperalgesia and allodynia. Advantages of the cold plate test are its relative speed and the ability for accurate temperature control.

Unlike the hot plate test, the cold plate test is particularly useful for models where only one paw is affected or sensitized by the experimental compound unilateral pain as guarding of the affected limb can be easily achieved, and thus can be easily quantified.

Quantification of more subtle behaviors, such as walking backwards or grooming of the front paws has thus been proposed as alternatives to quantify cold pain behaviors, although the validity of this approach has not been systematically assessed. The acetone evaporation test, first described in , is a technique used to measure aversive behaviors triggered by evaporative cooling and is typically considered as a measure of cold allodynia Carlton et al. Consistent application of acetone can be challenging, as acetone has a lower surface tension than water Figure 4.

Methods used to assess cold-evoked pain like behaviors and temperature preference in rodents. A Acetone evaporation test. Acetone is applied to the hind paw and the nocifensive response s is counted, timed or scored. B Cold plantar assay. A cold stimulus is applied to the hind paw using a cut off syringe filled with dry ice or wet ice and the time to paw withdrawal is recorded. Sensitivity to cold is recorded either by quantifying the number or duration of nocifensive responses, or scoring of the severity of the response e.

As the nocifensive response can be too fast for an investigator to quantify in real time, video recordings that are played back in slow motion may be required to accurately analyze the response to acetone. Nevertheless, the acetone evaporation test has been validated in multiple models of inflammatory and neuropathic pain in both mice and rats Choi et al.

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