WO1998044845A1

WO1998044845A1 - Apparatus and method for measuring neuromuscular function

Info

Publication number: WO1998044845A1
Application number: PCT/US1998/007423
Authority: WO
Inventors: Zuoshang Xu; Jiming Kong
Original assignee: University of Massachusetts Amherst
Current assignee: University of Massachusetts Amherst
Priority date: 1997-04-09
Filing date: 1998-04-09
Publication date: 1998-10-15
Anticipated expiration: 1999-10-09
Also published as: AU7114998A

Abstract

An apparatus for assessing muscle strength in a rodent, and methods of using the apparatus are disclosed. The apparatus comprises a rodent grip element (9, 10), an assembly including a motor (1), a disc (2), and a lever arm (3) connected to the rodent grip element (9, 10) for moving the rodent grip element (9, 10) at a controlled speed at a given distance above a surface. In operation a rodent is suspended from the rodent grip element (9, 10), and the length of time that the rodent remains suspended is measured. The length of time that a rodent remains suspended is indicative of the relative muscle strength of the rodent. Weak muscle strength in a rodent is indicative of a neuromuscular disease. Medicaments can be screened using the apparatus of the invention by assessing the muscle strength of the rodents after treatment with a potential medicament. The rodents serve as models for human neuromuscular diseases.

Description

APPARATUS AND METHOD FOR MEASURING NEUROMUSCULAR FUNCTION

Cross Reference To Related Applications This application claims priority from U.S. Provisional Application Serial No. 60/043,869 filed April 9, 1997.

Background of the Invention The invention relates to an apparatus and method for measuring neuromuscular function. The neuromuscular disorders are a major group of diseases causing muscle weakness, fatigue, paralysis, and in some cases, death arising from defects in the motor unit. A motor unit includes the motor neuron cell body, the motor neuron axon in the peripheral or cranial nerve, the neuromuscular junction, and the muscle fibers innervated by the motor neuron. A defect in any portion of the motor neuron results in denervation that causes shrinkage of the musculature and muscle fiber atrophy. Neuromuscular disorders include diseases such as amyotrophic lateral sclerosis, primary lateral sclerosis, spinal muscular atrophy, familial spastic paraparesis, multiple sclerosis, diabetic neuropath, myasthenia gravis, and Duchenne's muscular dystrophy.

Amyotrophic lateral sclerosis (ALS) is a common neuromuscular disorder. ALS is characterized by the progressive loss of both lower motor neurons and upper motor neurons, resulting in a loss of nerve function throughout the body. As denervation progresses with time, muscle atrophy progressively becomes worse, typically resulting in paralysis, and ultimately death. Although ALS effects both lower and upper neurons throughout the body, several specific neurons are unaffected in patients having ALS, such as neurons of the sensory system, neurons involved in the control of coordination of movement, neurons in the brain that are needed for cognition, neurons involved in ocular motility, and the parasympathetic neurons of the sacral spinal cord which innervate the sphincters of the bowel and the bladder.

Currently there are no treatments prescribed for ALS. Several potential therapeutic agents, such as ciliary neurotrophic factor, insulin-like growth factor- 1, and 2-amino-6 (trisluoromethoxy) benzothiazole or a salt thereof (U.S. Patent No. 5,527,814) have been proposed as being useful for treating ALS, but the therapeutic value of these drugs has not yet been established in humans. Identification of other potential therapeutics for the treatment of ALS would represent a significant advance in the field.

Recent research has indicated that a particular form of ALS, known as familial ALS (accounting for approximately 5 to 10% of all ALS cases) , is linked to mutations in a gene on chromosome 21 which encodes Cu/Zn- dependent superoxide dismutase (SODl) . Between 15 and 20% of familial ALS cases are believed to be caused primarily by a class of mutations in the SODl gene. The mutant SODl subunit apparently assumes a toxic property which results in neuronal death. Initially it was thought that the mutation caused a loss of SODl activity, which resulted in the neuronal death, but subsequent studies have indicated that loss of SODl activity is not relevant to the progression of ALS. To date, at least 45 different mutations in the SODl gene have been identified in ALS patients.

Transgenic mice expressing the SODl gene encoding various mutations have been prepared by many researchers. Transgenic mice expressing mutant neurofilament subunits which mimic the pathology of ALS seen in human disease have also been created. Although neurofilament mutations have not been shown to play a role in human disease, the transgenic mice serve as a useful model for the creation of symptoms 'associated with ALS. In particular, mutations in the neurofilament heavy gene (NF-H) and neurofilament light gene (NF-L) result in neuronal changes that mimic ALS.

These and other transgenic animals which serve as models of ALS are useful for screening potential therapeutics for the treatment of ALS. The effects of a particular therapeutic may be established by analyzing any physiological changes in the neurons and muscles of the animal which occur as a result of the administration of the therapeutic. Physiological changes in transgenic mice have been examined by a variety of measures, such as electrophysiological recordings (Azzouz et al . , Muscle and Nerve, v. 20, p . 45-51 , 1997) , direct tissue analysis (Chiu et al . , Molecular and Cellular Neuroscience, v. 6 p . 349-362, '1995) , behavioral changes such as shaking or tremors in limbs, (Chiu et al . ) , weight loss, shortening of stride length, and progressive loss of muscle strength (Collard and Julian, J. Psychiatr. Neurosci . , v. 20, p . 80-86, 1995) .

Methods for assessing muscle strength are particularly useful for analyzing the effects of therapeutics on animal models of ALS because large numbers of therapeutics may be screened at a time. The muscle strength test described by Collard and Julian consists of a pencil which is held above a surface for a mouse to grasp. The length of time which a mouse is capable of grasping the pencil is said to be predictive of the muscle strength of the mouse. A test called the "Coat Hanger Test," described by Escorihuela et al . (Escorihuela et al . , Neuroscience Letters, v. 199, p . 143 -146, 1995) consists of a coat hanger in which a mouse is allowed to grasp the middle of the coat hanger wire. Again the amount of time that the mouse is able to remain suspended from the wire is measured and is said to be predictive of the muscle strength of the mouse. Grip strength tests described by Mitsumoto et al . (Mitsumoto, Science, v. 265, p . 1107-1110, 1994) are also useful for measuring muscle strength. The grip strength test involves a process of holding a mouse in the air and causing the .mouse to grasp a horizontal wire strain gauge with both forelimbs. The horizontal wire strain gauge is connected to a dynamometer for measuring muscle strength. Each of these muscle strength tests measure the forelimb strength of a rodent, and have been used in research studies for this purpose for many years.

Summary of the Invention

The invention features an apparatus for measuring muscle strength or deterioration thereof in rodents, e.g., rodent models of human neuromuscular diseases.' The apparatus includes a rodent grip element which moves at a controlled speed above a surface. Muscle strength can be assessed by measuring the length of time a rodent can grasp the moving rodent grip element before falling onto the surface .below. Rodents with neuromuscular problems are only able to grasp the moving rodent grip element for a relatively short period of time (e.g., a few seconds), whereas normal rodents or rodents with neuromuscular problems which have been successfully treated with medicaments are able to grasp the moving rodent grip element for longer periods of time (e.g., more than two minutes on average) . Potential medicaments for human neuromuscular diseases can easily be screened using the apparatus of the invention by observing the effects of the medicaments on the muscle strength of the rodents having conditions that model human disease.

The apparatus of the present invention has numerous advantages over the prior art devices used for assessing muscle strength in rodent models. The prior art devices include a stationary rod or loop which a rodent grasps and is suspended from until falling to the surface below, presumably once the muscles of the rodent are fatigued. It was discovered that on some occasions a rodent will release its grip on the stationary rod or loop even before the muscles of the rodent became fatigued, causing unpredictable and inaccurate results. The present invention is based on the finding that by providing a moving rodent grip element for the rodent to grasp, the rodent becomes disoriented and fearful and does not randomly drop to the surface until the muscles of the rodent actually are fatigued. Therefore, the present invention provides an apparatus which is useful for screening medicaments that affect muscle strength and that yields more accurate results than prior devices.

A further advantage of the new apparatus is that it permits the assessment of muscular strength in both the fore- and hind-limbs. The prior art devices consisted of a horizontal wire or other type of rod which the rodent grasped with its forelimbs. In some cases the rodent was allowed to grasp the horizontal wire with the hindlimbs, but this technique generally fails because the mouse will quickly release its grasp or grip the wire with its fore-paws. In one embodiment of the present invention, the apparatus includes a vertical wire or rod for the rodent to grasp. It was discovered that a rodent will grasp a vertical wire or rod with both the fore- and hind-limbs. Therefore, the apparatus of the present invention is useful for assessing strength of both the fore- and hind-limbs. In one aspect the invention features an apparatus for assessing relative muscle strength in a rodent . The apparatus includes a base, a grip element support extending substantially vertically from the base, at least one rodent grip element movably attached to the grip element support, and a motor operably connected to the rodent grip element for moving the rodent grip element at a controlled speed above a surface . The surface is a rodent catching device. When the rodent releases its grasp on the rodent grip element, the rodent drops onto the rodent catching device. Optionally the surface also may serve as a support for the base. The surface which supports the base, however, may be a different surface than the surface which is the rodent catching device.

In another embodiment, the apparatus includes at least one rodent grip element and a mechanism for moving the rodent grip element at a controlled speed above a surface according to a predetermined pathway, wherein a minimum distance between the rodent grip element and the surface is at least 1.5 feet and wherein a maximum distance between the rodent grip element and the surface is 6 feet. According to one embodiment, the rodent grip element is a bar, e.g., in the form of a rod or a loop. In a preferred embodiment the rodent grip element is a vertical wire rod. Optionally, the apparatus may also include a stop positioned above the rodent grip element to prevent the rodent from climbing onto other parts of the apparatus. In a preferred embodiment the stop is a platform.

The invention further features an apparatus that includes at least one rodent grip element and a mechanism for moving the rodent grip element at a controlled speed above a surface according to a predetermined pathway. The mechanism for moving the rodent grip element is a disk-motor assembly including a motor which is operably connected to a disk such that the motor moves the disk and a rod having a first end connected to the disk and a second end connected to the rodent grip element such that movement of the disk causes the rod and the rodent grip element to move.

In another embodiment, the invention features an apparatus including at least one rodent grip element, wherein the rodent grip element is a bar in the shape of a rod having a length of less than 7 inches or a loop having a diameter of less than 7 inches, and a mechanism for moving the rodent grip element at a controlled speed above a surface according to a predetermined pathway. In one embodiment, the rodent grip element is a loop having a diameter of less than 4 inches. In another embodiment, the rodent grip element is a rod having a length of less than 4 inches .

Other embodiments of the invention may include one or more of the following features. The apparatus can include a stop. The stop can be positioned above the rodent grip element . In a preferred embodiment, the stop is a platform. The apparatus can include a sensor for detecting movement. The sensor can be positioned between the rodent grip element and the surface and is capable of detecting the movement of a rodent as it drops from the rodent grip element . The rodent grip element can be a disk/lever-motor assembly. The disk/lever-motor assembly optionally can include a motor which is operably connected to a disk or lever. As the motor moves the disk or lever and a rod having a first end connected to the disk or lever and a second end connected to the rodent grip element causes the rod and the rodent grip element to move. The apparatus can also include a mechanism for positioning the rodent grip element above a surface. The mechanism can be any type of element which is capable of supporting the rodent grip element above the surface. For example, the mechanism may be the disk/lever-motor assembly discussed herein. The apparatus can also include a stabilization element attached to the rodent grip element .

The rodent grip element can also include a grip element with both a fore-grip element and a hind-grip element. Preferably the grip element with both a fore- grip element and a hind-grip element is a vertical wire. Various element or combinations of elements can be included in each apparatus of the invention. For instance each apparatus may include a mechanism for moving the rodent grip element which is a disk/lever- - motor assembly, which optionally may include a motor operably connected to a disk or lever such that the motor moves the disk or lever and a rod having a first end connected to the disk or lever and a second end connected to the rodent grip element such that movement of the disk or lever causes the rod and the rodent grip element to move; a mechanism for positioning the rodent grip element above a surface; a stabilization element; a stop; a grip element with both a fore-grip element and a hind-grip element; or a sensor for detecting movement or any combination of elements thereof. In another aspect, the invention features a method for assessing muscle strength of a rodent. The method includes the steps of providing a rodent grip element suspended above a surface, causing a rodent to grasp the rodent grip element, moving the rodent grip element at a controlled speed, and measuring a length of time that the rodent remains suspended from the rodent grip element . In one embodiment, the rodent is a transgenic rodent expressing an amyotrophic lateral sclerosis phenotype . According to one embodiment of the method, the rodent grip element is rotated at a controlled speed, e.g., of at least 10 or 20 rpm.

In to another aspect, the invention features a method for screening a medicament to determine if the medicament affects muscle strength of a rodent . The method includes administering a medicament to a rodent, causing the rodent to grasp a moving rodent grip element, and measuring a length of time that the rodent remains suspended from the rodent grip element, wherein the length of time being indicative of the ability of the medicament to influence muscle strength.

In different embodiments, the rodent exhibits symptoms of muscular dysfunction and the medicament is screened to determine if the medicament improves muscle strength of the rodent or prevents further deterioration of muscle strength of the rodent . The rodent can be a transgenic rodent expressing an amyotrophic lateral sclerosis phenotype, caused by a mutant Cu/Zn superoxide dismutase genotype.

The rodent can also be susceptible to age dependent muscle loss and the medicament can be screened to determine if the medicament delays the onset of muscle deterioration. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims .

Brief Description of the Drawings Fig..1 is a schematic side view of an apparatus and methods of use thereof of the invention. Fig. 2a is a graph showing the average hanging time for a transgenic mouse expressing the mutant SODl (G93A) gene.

Fig. 2b is a graph showing the average hanging time of the transgenic mice expressing normal SODl gene. Fig. 2c is a graph showing the average hanging time of wild type mice.

Fig. 3A is a graph showing the age-dependent decline of muscle strength in G93A mice.

Fig. 3B is a graph showing the highly variable onset of muscle weakness among different G93A mice.

Fig. 3C is a graph showing a synchronized plot of muscle strength decline in individual G93A mice.

Fig..3D is a graph showing the average time course of muscle strength decline in G93A mice.

Detailed Description

The present invention involves an apparatus and methods of use thereof for assessing the muscular strength or deterioration thereof in a rodent model of human neuromuscular disease. The apparatus can be used to determine whether a particular rodent, such as a transgenic rodent expressing a mutated gene potentially associated with a neuromuscular disease, develops a neuromuscular phenotype resulting in a progressive deterioration of muscle strength. The apparatus can also be used to identify or evaluate drugs, pharmaceuticals, treatments, and intervention regimes for the prevention or treatment of neuromuscular diseases and disorders involving muscle deterioration or destruction, and to screen agents which can cause or exacerbate the progression of such disorders.

The apparatus of the invention includes at least one rodent grip element and a mechanism for moving the rodent grip element at a controlled speed above a stationary surface.

A "rodent grip element" is a physical structure capable of supporting the weight of a rodent and having a shape which .allows a rodent to grasp onto it. The rodent grip element can be any shape that allows it to be grasped by a rodent . For example the rodent grip element may be a straight rod, a concave or convex rod, a rod having multiple bends or twists, a loop, or a bar or planar surface with a channel or other form of depression along at least one edge of the bar or planar surface.

The rodent grip element may be composed of two separate pieces, which may optionally be joined to one another. For instance, the rodent grip element may consist of two loops which are positioned next to one another, such that the rodent can grasp each loop with a separate limb. The loops may be connected to one another to maintain the loops within a desirable distance of one another and/or to hold the loops in a particular orientation.

The rodent grip element may be oriented in a horizontal or vertical position. When the rodent grip element is oriented in a horizontal position, the apparatus is more typically useful for assessing the forelimb strength of a rodent, because in such embodiments the rodent typically grasps the rodent grip element with the two forelimbs. When the rodent grip element is oriented in a vertical position, the apparatus is more typically useful for assessing the fore- and hind-limb strength of the rodent, because when the rodent grip element is in a vertical position the rodent tends to grasp the element with both sets of limbs. The vertical orientation is particularly useful for assessing muscle strength in rodents expressing an ALS phenotype because ALS causes weakness in hind-limbs prior to causing weakness in the forelimbs. Alternatively, when the rodent grip element is oriented in a vertical position the rodent grip element may include a stabilization element which enables the rodent to stabilize its balance on the vertical rodent grip element. For example, when the rodent grip element is a vertical rod the stabilizing element may be a bar or loop which is positioned below the vertical rod to prevent the rodent from sliding off the vertical bar.

The rodent grip element may be made of any material capable of supporting the weight of a rodent. Materials which are useful for forming the rodent grip element according to the invention include but are not limited to metal, plastic, wire, fabric, rubber, wood, or string. Preferably, in some embodiments, the rodent grip element is made from a wire material. Although the rodent grip element can be any size, it is anticipated that a rodent grip element which is excessive in size will not provide any advantages to the operation of the apparatus but may make the apparatus more cumbersome to operate. For this reason, typically, the rodent grip element has a size which does not exceed the length of the rodent being studied. For instance, when the rodent grip element is a rod, the rodent grip element can have a length of less than 7 inches. Typically, the rod has a length of less than 4 inches. When the rodent grip element is a loop, the rodent grip element can have a diameter of less than 7 inches. Typically, the loop has a diameter of less than 4 inches.

The apparatus also includes a mechanism for moving the rodent grip element at a controlled speed above the stationary surface. A "mechanism for moving the rodent grip element" as used herein refers to any mechanism capable of moving the rodent grip element according to a predetermined pathway. The mechanism for moving the rodent grip -element can be any source that generates movement. Sources which generate movement include, but are not limited to, a motor, a winding mechanism, a wind driven mechanism, a water driven mechanism, and a nuclear power driven mechanism. Typically, the mechanism for moving the rodent grip element is a motor. According to one embodiment of the invention, the mechanism for moving the rodent grip element is a mechanism that moves the rodent grip element in a vertical plane. This embodiment is depicted in the form of a disk/lever-motor assembly in Fig. 1. A motor (1) positioned on a horizontal surface causes a disc (2) which is positioned in a plane perpendicular to the horizontal surface, to rotate. An arm (3) having a first end (4) fixedly attached to the disc (2) is moved in a circular or elliptical path as the disc rotates. The arm (3) has a second end (5) which includes a bearing (6) having a central track. A suspension assembly (7) having a ring (8) on a first end, a wire loop (10) on a second end, and a suspension cable (9) connecting the ring (8) and the wire loop (10) , is positioned with the ring (8) in the track of the bearing (6) . The suspension cable (9) is suspended in a plane perpendicular to the horizontal surface. As the disc (2) rotates, the wire loop (10) is transported in a vertical plane along a circular or elliptical path. The apparatus functions when the rodent grasps the suspension cable (9) (measure fore- and hind-limb strength) or the wire loop (10) (measure forelimb strength) , and is rotated in the vertical plane. Typically, the assembly includes a stop

(11) to prevent the rodent from climbing from the wire loop onto the other pieces of the assembly. The mechanism for moving the rodent grip element can also be a mechanism for moving the rodent grip element in a horizontal plane. For example, the mechanism can be a disk/lever motor assembly which moves the rodent grip element in a horizontal plane around a central axis. The central axis can be a point at the center of a circle in which the rodent grip element moves around the perimeter of the circle. Alternatively, the central axis can be a point on a line which the rodent grip element traverses. The mechanism for moving the rodent grip element, alternatively, can be a mechanism for moving the rodent grip element in some combination of a vertical and horizontal plane. For example, the rodent grip element can be moved along a U-shaped path which involves both vertical and horizontal movement. The actual mechanism responsible for moving the rodent grip element can be any device known in the art .

The rodent grip element is moved at a controlled speed in order to provide a consistent fluid movement. A consistent and fluid movement enhances the stability with which the rodent is capable of grasping the rodent grip element. The controlled speed can be constant or smoothly varied as long as the movement is fluid and not inconsistent or jerky. Inconsistent or jerky movements resulting from changes in the speed of movement can influence the ability of the rodent to grasp the rodent grip element causing the rodent to prematurely release the rodent grip element and thus interfere with the correlation between the length of time that the rodent can grasp the rodent grip element and the muscle strength of the rodent .

The actual speed with which the rodent grip element moves depends on a number of factors including the range of movement of the rodent grip element and the type of mechanism causing that movement . The rodent grip element, as shown in Fig. 1, is rotated along a circular or elliptical pathway by the movement of the disk or lever (2) . When the disk (2) has a radius of approximately 6 inches, the minimum and maximum speeds useful according to the invention are 10 rpm and 45 rpm, respectively. A typical speed range for this embodiment is approximately 20-30 rpm, e.g., 24 or 25 rpm.

Although the rodent grip element of the apparatus moves through a predetermined pathway, it is desirable that the rodent grip element in general maintains an overall consistent orientation. For example, when the rodent grip element is a horizontally disposed straight cylindrical -rod, it is desirable that the rod is maintained in that horizontal orientation.

The rodent grip element must be suspended above a surface. The surface, which can be solid or liquid, functions to catch the rodent when the rodent releases its grasp of the rodent grip element . When the surface is a solid horizontal stationary surface, the surface can also function to maintain the apparatus of the invention in a fixed position. Typically, the surface is a horizontal stationary surface.

The rodent grip element must be suspended far enough above the surface when the rodent grip element is positioned at the point closest to the surface, to prevent the rodent from touching the surface. Additionally, if the rodent grip element is positioned too closely -to the surface, the rodent will release the rodent grip element prematurely before the muscles of the rodent are fatigued. As long as the rodent grip element is suspended a minimum distance above the surface the actual distance between the rodent grip element and the surface does not influence or cause the rodent to prematurely release its grasp on the rodent grip element prior to experiencing muscle fatigue, in an apparatus in which the rodent grip element is moving. The minimum distance according to the invention is more than one foot, typically, is at least one and one half feet, or alternatively, is at least two feet. Although there is no maximum distance between the support and the surface which would effect the results of the study, for ethical reasons, the rodent grip element should not be suspended higher than that distance that would cause injury to the rodent when it falls to the surface. The maximum distance that will not cause injury to a rodent, of course, depends on the type of surface on which the rodent lands, and the type of rodent.

The apparatus of the invention also can include a means for preventing a rodent from climbing from the rodent grip element to other regions of the apparatus . Typically, a stop or other means for blocking the rodent may be positioned above the rodent grip element to prevent the -rodent from climbing onto other regions of the apparatus. The stop should be large enough that the rodent cannot grasp the edges of the stop with its free limbs. The actual size of the stop depends on the size of the rodent used in the study. Typically, the stop is a platform. Muscle strength is assessed by measuring the length of time that the rodent is capable of grasping the rodent grip element before falling to the surface, the length of time being indicative of the muscle strength. Muscle strength can be evaluated in several different ways. A progressive loss of muscle strength of a particular rodent can be evaluated by assessing, at particular intervals such as every 10 days, the length of time that the mouse can grasp the rodent grip element (the hanging time) . A profile depicting the progressive decline in muscle strength is determined by plotting the hanging time for the particular mouse as a function of time (age) . Whether a medicament can prevent the onset of muscle deterioration, maintain muscle strength, or reverse the loss of muscle strength by improving muscle function can also be assessed using the apparatus of the invention. Rodents which develop age dependent muscle deterioration are administered the medicament at various times depending on whether it is desirable to prevent the onset of deterioration, maintain or improve muscle strength. Muscle strength is evaluated by assessing the hanging time in comparison to the average hanging time for mice of that particular strain and/or in comparison to the hanging time prior to administration of the medicament . The length of time that the rodent is capable of grasping the rodent grip element can be measured by any means known in the art. For example, the suspension time can be measured with a manual instrument such as a stopwatch and manually recorded into a log for subsequent data comparison. Alternatively, the suspension time can be measured and optionally recorded by a mechanical or electrical sensor. Typically, the apparatus includes a sensor for detecting the time which the rodent releases its grasp on the rodent grip element and falls to the surface. Optionally the sensor can include a recording means which records the time value . Sensors have been described extensively in the art, for example in US Patent Nos . 5,228,019, 5,553,857, 5,430,953 and, Stδff et al . , Psychopharmacology, V. 80 , P . 319 -324 , 1983 . The sensor also can be a means which detects movement, such as that of a falling rodent passing a detection beam. Alternatively the sensor can be a means which detects pressure, such as a mechanism in the surface which detects the ^'falling rodent. Additionally the sensor can be a means that detects the release of a grip or the loss of a weight, such as when the rodent releases the rodent grip element .

The invention also encompasses a method for assessing muscle strength of a rodent by providing a rodent grip element suspended above a surface, moving the rodent grip element and causing a rodent to grasp the moving rodent grip element . The muscle strength of the rodent is assessed by measuring a length of time that the rodent remains suspended from the rodent grip element as discussed above.

The method is useful for any purpose that it is desirable to asses muscle strength in rodents. For example, the method of the invention can be useful for assessing the muscle strength of a transgenic rodent in order to determine whether the transgenic rodent is useful as a model of neuromuscular disease involving muscular deterioration. The method can also be useful for screening medicaments which influence muscle strength or deterioration . The invention also includes encompass a method for screening a medicament to determine if the medicament improves muscle strength of a rodent having impaired muscle strength. The method involves administering a medicament to a rodent exhibiting symptoms of muscular dysfunction, causing the rodent to grasp a moving rodent grip element and measuring a length of time that the rodent remains suspended from the rodent grip element . A determination of whether the medicament, alters the muscle strength of the rodent can be carried out by comparing the length of time that a rodent which received the medicament grasps the rodent grip element with the length of time that a comparable control rodent which, did not receive the medicament grasps the rodent grip element. A comparable control rodent is a rodent of the same strain as the test rodent. For example, when the test rodent is a transgenic mouse having a mutant SODl gene such as the G93A strain, a comparable control rodent is a transgenic mouse of the G93A strain. When the muscle strength of the transgenic rodent varies as the rodent ages it is important that the comparable control rodent also is at the approximately same point of development as the test rodent .

A "rodent" is a small mammal of the species rodentia having limbs and claws which allow the animal to hang from a rodent grip element. Rodents that can be used according to the invention include, for example, mice, rats, squirrels, hamsters, and gerbils.

The invention is useful for studying any rodent which exhibits symptoms of muscle dysfunction or any rodent susceptible to age related muscle loss. A "rodent exhibiting symptoms of muscular dysfunction" is a rodent that has demonstrate symptoms of muscle loss or deterioration. Symptoms of muscular dysfunction may be analyzed by -any method known in the art. For example, muscular dysfunction may be assessed by examining by a variety of measures, such as electrophysiological recordings (Azzouz el al . Muscle and Nerve, v. 20, p . 45-51 , 1997) , direct tissue analysis (Chiu et al . , Molecular and Cellular Neuroscience, v. 6. p . 349-362, 1995) , behavioral changes such as shaking or tremors in limbs, (Chiu et al . ) , weight loss, shortening of stride length and progressive loss of muscle strength (Collard and Julian, J Psychiatr. Neurosci . , v. 20, p . 80 -86, 1995) . In one embodiment of the invention the rodent exhibiting symptoms of muscular dysfunction is a transgenic rodent expressing an amyotrophic lateral sclerosis phenotype. Such transgenic rodents are well known in the art and include for example a transgenic rodent expressing a mutant Cu/Zn superoxide dismutase genotype which has progressed to an age ( approximately 137 days) when muscle deterioration begins.

A rodent susceptible to age related muscle loss is a rodent which will develop a muscular dysfunction phenotype. Such rodents include but are not limited to the transgenic rodents expressing genes having mutations in the SODl gene or in the NF-H or NF-L genes.

Examples Example 1 ; Muscle Strength Measurements

Three groups of mice were obtained for the study, a transgenic mouse expressing the mutant SODl (G93A) gene (obtained from Jackson Laboratories, Acadia National Park, ME and bred at the Worcester Foundation, Shrewsbury, MA) , transgenic mice expressing normal SODl gene (obtained from Jackson Laboratories, Acadia National Park, ME and bred at the Worcester Foundation, Shrewsbury, MA) , and wild type mice (Worcester Foundation, Shrewsbury, MA) .

The mice were allowed to grab onto a vertical wire (3 mm in diameter) with a small loop at the lower end of an apparatus as shown in Figure 1. The wire was maintained in a vertically oriented circular motion (radius: 10 cm) at 24 rpm. The time at which each mouse dropped from the wire was recorded with a stopwatch and used as a measure of neuromuscular function. The results are presented in Figs. 2A-2C in terms of the length of time in seconds that the mice are capable of hanging onto the wire as a function of the age of the mouse. As demonstrated in Figure 2a, the mice expressing the mutant SODl gene experiences a significant loss of muscle strength at the age of approximately 137 days compared to mice expressing the normal gene and wild type mice, demonstrating that these mice experience age dependent progression of muscle weakness.

Example 2 ; Screening Medicaments

Four groups of mice are used in the study, two transgenic mice expressing the mutant 30 SODl (G93A) gene, a transgenic mouse expressing normal SODl gene, and a wild type mouse, each 160 days old. One of the G93A mice is pretreated with a test medicament . The muscle strength of each of the four mice is then assessed as disclosed above in Example 1. If the test medicament improves the muscle strength of the treated G93A mouse relative to the untreated G93A mouse, then the test medicament may be useful for treating ALS.

Example 3 : An Assay for Measuring Muscle Strength

A simple and objective assay to measure muscle strength was developed to follow ALS disease progression in G93A mice. In this assay, the time that a mouse was able to hang onto a wire was measured as an indication of muscle strength.

Mice transgenic for the mutated human SODl G93A (TgN[SODl-G93A] IGur) and wild type human SODl (TgN[S0Dl] 2Gur) were purchased from The Jackson Laboratory and bred in the University of Massachusetts

Medical School animal facility.

A muscle strength test was conducted using a device as shown in Fig. 1. Mice were allowed to grab onto a vertical wire (2 mm in diameter) with a small loop at the lower end. A vertical wire allows mice to use both fore- and hind-limbs to grab onto the wire. Although in the first few tests some mice used forelimbs predominantly, they usually learn to use all four limbs after a few trials. This results in a significant improvement during the first three trials, after which the performance stabilized. Thus, both the fore- and hind- limbs contribute to the measured muscle strength in this assay. The wire was maintained in a vertically oriented circular motion (the circle radius was 10 cm) at 24 rpm. The time that the mouse was able to hang onto the wire was recorded with a timer. Because most mice fell within 5 minutes, testing was cut off at 300 seconds to test more animals in a limited time period. Mice were usually tested once a week starting from 90 days old and continued until they could no longer hang onto the wire. Test results of mice in different age groups revealed an age-dependent decline of hanging time for G93A mice, but not for wild type mice (WT) and wild type human SODl (WS) transgenic mice. See Fig. 3A, which shows the time that mice were capable of hanging onto a wire. This time is used as an indication of muscle strength. To determine how muscle weakness develops in individual animals, G93A and wild type animals were tested once a week beginning at 90 days of age and continuing until the G93A mice could no longer hold onto the wire. The results revealed several interesting aspects of muscle strength change. First, both the onset and the duration of muscle strength decline were highly variable among different individuals, ranging from age 113 days to 188 days for the former and 35 to 94 days for the latter. As shown in Fig. 3B, the onset of muscle weakness is highly variable among diffferent G93A mice.

Second, the time course of muscle strength change apparently went through four different stages. As shown in Figs. 3C and 3D, there is a pre-muscle weakness stage (PMW) , during which muscle strength was maintained at the normal level; a rapid declining stage (RD) , during which the hanging time declined sharply (usually by more than 50% within a period of two weeks) ; a slow declining stage (SD) , which followed the RD stage and lasted for 4 to 11 weeks; and a paralysis stage (Para), during which paralysis of limbs began and the mouse could no longer hold onto the wire.

Each stage in the progression of ALS in G93A mice were correlated with specific pathological features. The PMW stage (just prior to the RD stage) shows little difference from the wild type under a light microscope. However, abundant mitochondrial abnormalities can be found by electron microscopy. The onset of muscle weakness (RD stage) correlates with a massive mitochondrial vacuolation, signifying the beginning of a degenerative process in motor neurons that compromises their function. The SD stage correlates with axonal atrophy and a gradual loss of motor neurons. However, the majority of motor neurons are alive until the very end stage of the disease. Several implications can be drawn from these results. First, abundant mitochondrial abnormalities are the most prominent pathological feature prior to the onset of muscle strength decline. In addition, the onset of a sharp muscle strength decline is correlated with a massive mitochondrial vacuolation. Second, the vacuolation is a transient process. It correlates with the onset of muscle weakness, but decreases towards the end stage of the disease. Third, the initial muscle weakness signifies a stage in which the degenerative process in large motor neurons begins to compromise their function. It does not, however, represent a massive loss (or death) of these neurons. The loss of the majority of motor neurons does not occur until the paralysis stage, the end stage of the disease. The presence of the majority of motor axons during the RD and SD stages indicates that even after the muscle weakness begins, most motor neurons have not died, and thus could be rescued by effective therapeutic intervention.

The current study suggests that mitochondrial damage is at the center of the degeneration mechanism. The assay described in this example for ALS disease progression sets the stage for further investigations of the disease mechanism in animal models. This assay enables the study of animals with defined disease stages, thus allowing meaningful comparisons of pathological evolution among different SODl mutations. By grouping mice according to their disease stages, inconsistencies caused by heterogeneous disease stages can be avoided, therefore allowing a full construction of the sequence of events (including both biochemical and morphological events) leading to motor neuron death.

The assay also provides a useful means to evaluate the effectiveness of potential therapeutic agents. For example, the assay can be repeated with therapeutic agents administered at different stages of muscle strength change. Results from the assay can be used to determine whether the therapeutic agent has an affect on a particular stage. Alternatively, the therapeutic agent can be administered at the beginning of the assay and the results used to determine if the agent affects any, one, or more of the stages.

Other Embodiments The apparatus, as described above, also can include a second grip element added, e.g., to disk 2 onto a horizontal member (like 3) but opposite, i.e., across the center of the disk, from arm 3.

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantageous, and modifications are within the scope of the following claims.

What is claimed is:

Claims

1. An apparatus for assessing relative muscle strength in a rodent, comprising: at least one rodent grip element; and a mechanism operably connected to the rodent grip element for moving the rodent grip element at a controlled speed at a given distance above a surface.

2. The apparatus of claim 1, wherein the given distance between the rodent grip element and the surface is at least 1.5 feet, and wherein a maximum distance between the rodent grip assembly and the surface is 6 feet .

3. The apparatus of claim 1, wherein the mechanism for moving the rodent grip element is a disk/lever-motor assembly including a motor which is operably connected to a disk or lever such that the motor moves the disk or lever, and a rod having a first end connected to the disk or lever and a second end connected to the rodent grip element such that movement of the disk or lever causes the rod and the rodent grip element to move.

4. The apparatus of claim 3 , wherein the rodent grip element is a bar having a shape selected from the group consisting of a rod having a length of less than 7 inches and a loop having a diameter of less than 7 inches.

5. The apparatus of claim 4, wherein the rodent grip element is a loop having a diameter of less than 4 inches .

6. The apparatus of claim 4 , wherein the rodent grip element is a rod having a length of less than 4 inches.

7. The apparatus of claim 1, further comprising a stop positioned above operatively connected to the rodent grip element .

8. The apparatus of claim 1, further comprising a sensor attached to the apparatus for detecting movement, wherein the sensor is positioned between the rodent grip element and the surface and wherein the sensor detects the movement of a rodent as it drops from the rodent grip element.

9. An apparatus for assessing relative muscle strength in a rodent, comprising: a base; a grip element support connected to the base; at least one rodent grip element movably attached to the grip element support ; and a motor operably connected to the rodent grip element for moving the rodent grip element at a controlled speed at a given distance above a surface.

10. The apparatus of claim 9, further comprising a stabilization element attached to the rodent grip element .

11. 'A method for assessing muscle strength of a rodent, the method comprising: providing a rodent grip element suspended above a surface; causing a rodent to grasp the rodent grip element; moving the rodent grip element at a controlled speed; and, measuring a length of time that the rodent remains suspended from the rodent grip element .

12. The method of claim 11, wherein the rodent grip element is connected to a substantially circular disk rotated at a speed of at least 10 rpm.

13. The method of claim 11, wherein the rodent grip element is rotated at a speed of at least 20 rpm.

14. The method of claim 11, wherein the rodent is a transgenic rodent expressing an amyotrophic lateral sclerosis phenotype.

15. A method for screening a medicament to determine if the medicament affects muscle strength of a rodent, the method comprising: administering a medicament to a rodent; causing the rodent to grasp a moving rodent grip element; and, measuring a length of time that the rodent remains suspended from the rodent grip element, the length of time being indicative of the ability of the medicament to influence muscle strength.

16. The method of claim 15, wherein the rodent is a rodent exhibiting symptoms of muscular dysfunction and the medicament is screened to determine if the medicament improves muscle strength of the rodent .

17. The method of claim 16, wherein the rodent is a transgenic rodent expressing an amyotrophic lateral sclerosis phenotype.

18. The method of claim 17, wherein the transgenic rodent expressing an amyotrophic lateral sclerosis phenotype expresses a mutant Cu/Zn superoxide dismutase genotype.

19. The method of claim 15, wherein the rodent is a rodent exhibiting symptoms of muscular dysfunction and the medicament is screened to determine if the medicament prevents further deterioration of muscle strength of the rodent .

20. The method of claim 15, wherein the rodent is a rodent susceptible to age dependent muscle loss and the medicament is screened to determine if the medicament delays onset of muscle deterioration in the rodent.