2001.02.01 Simplified Technique of Measuring Foot Prints in Rats
Simplified Technique of Measuring Foot Prints in Rats and Current Status of Nerve Regeneration Research
Pramod K. Nelluri, MD*, Darryl W Peterson MD+
* From the Division of Plastic and Reconstructive Surgery, University of Texas, Health Science Center at San Antonio, Texas, and the + Hand & Microsurgery Unit, Louisiana Orthopedic Institute, Baton Rouge, Louisiana.
In the past decade, interest in nerve regeneration has shifted from the technology of surgical repair to the understanding of the neurobiology focussing on the nerve growth factors. FK 506, widely used as an immunosuppressant was recently shown to significantly enhance axonal growth in experimental animals. However, due to its severe nephrotoxic potential when used systemically, additional studies are warranted evaluating the risk/benefit ratios. Most studies determining the effects of these agents in enhancing nerve regeneration utilize the sciatic functional index (SFI) to analyze the functional recovery in rat models. In our experiment, we simplified the technique of walking track methods by using INDIA ink which was found to be simple, inexpensive and reasonably reliable in producing the foot prints. In addition, this report reviews some of the relevant nerve regeneration research in the past decade to give the reader familiarity with the current information regarding the various surgical techniques developed in terms of nerve repair and/or in a nerve gap.
Although the precise incidence of peripheral nerve injury is unknown, it is one of the most common traumatic injuries of the upper extremities. Studies on war victims (soldiers and civilians alike) have shown that peripheral nerve injuries account to about 70% –80% of all types of trauma (1,2). Advances in surgical technique and better understanding of nerve regeneration have greatly improved the functional recovery in such devastating injuries. Nontraumatic microtechnique, minimum amount of foreign material (suture) and avoidance of tension are some of the techniques developed over the years to yield the best outcome (3). In the past decade, focus in neurobiology research has been to develop an ideal nerve growth factor which enhances axonal growth in terms of the rate of advancement and as well as the density of nerve fibers. Various growth factors such as nerve growth factor (NGF), laminin, cortocotrophin (ACTH) and FK 506 have been shown to enhance nerve regeneration in experimental models (4-7). Assessment of functional recovery in animals is done by the measurements of foot prints of walking animal. Sciatic functional index (SFI) is a sensitive index of measurement in a rat model (8). This paper describes a simplified technique of the foot print measurements in rats using INDIA ink. In addition, we have reviewed the current literature in the field of peripheral nerve injury/nerve gap and ongoing nerve regeneration research.
Four young Lewis rats weighing 200 to 250 gms were used as experimental animals. For comparison, age matched controls were examined. All the animals in the experimental group were anesthetized and the left sciatic nerve was crushed at the level of the trifurcation point. Crush injury was carried out by a straight forceps for a duration of 60 seconds.
Functional Assessment: The animals were assessed on a daily basis with respect to their ability to move and walk. The post surgical day on which the animals began to move their right hind leg was recorded as ONSET of function. Similarly, the post surgical day on which the animal can move their right foot was recorded as WALKING function (9). At 3 and 4 weeks, walking track analysis was done using a simplified version of de Medinaceli technique (10). Briefly, india ink (Sanford Bellwood, Ilinois, USA) was used to mark the foot prints of the animals on a plain white paper. Measurements were made directly on the paper sheath and adjusted to the nearest mm. The total length of the foot was termed as print length factor (PLF), distance from the center of the first toe to the center of the fifth toe was termed as total toe spreading (TTS) and the distance between the center of the two adjacent digits was termed as intermediary toe spreading (ITS). For each animal, measurements were obtained from three foot prints and mean values from all digits averaged to give a single value.
Clinical signs of recovery in the hind feet and toes were manifested at around 3 weeks in all the animals. The foot and all toes were clearly discernible in the print both in normal and experimental animals. The normal animals mainly walk on their toes whereas the rats with nerve damage dragged their feet at 2 weeks and showed increased PL at 3 to 4 weeks when compared to controls (Fig 2, Fig 3). Further at 3 weeks, the fifth digit of the imprint is absent in experimental animals indicating continued deficit. Similarly, the total and intermediary toe spread distances were clearly reduced in the experimental rats when compared to the normal rats (Fig2, Fig 3). The sciatic functional index (SFI) was not done in this experiment as this study was mainly done to evaluate the feasibility of an alternate method of marking the foot prints.
In the face of a peripheral nerve injury, the distal nerve segment will undergo Wallerian degeneration while the proximal segment and cell body will respond with increased metabolic functions in preparation for regeneration. The axons at the site of injury will sprout regenerating units which must bridge the gap created by trauma or disease. Within one day of injury, the gap will be filled with exudate from the proximal and distal nerve stumps, including factors essential for successful axonal regeneration. Functional recovery from a nerve injury is studied by various methods such as electrophysiology, Tinel’s sign, pinch test, and histology. In animals, the function of regeneration is assessed by walking track methods like sciatic functional index (SFI). Orginally described in 1982 by De Medinacelli et al, it has been widely used as a standard method to assess motor recovery in a rat model (10). Briefly, this method utilizes a x-ray developer and an underdeveloped x-ray film to mark the foot prints of the animal. Recently, Gold et al simplified the technique by using tempora paint on a sheet of plain paper (7). In our experiment, we have used black india ink, a normal waterproof drawing ink widely available and inexpensive. The technique was found to be simple, and the prints were clearly discernible for measurements. The only disadvantage was that the animals had to be inked regularly after they have walked two sheets of paper. Increased PL in the recovering animals indicated that a paralyzed limb places the whole foot on the ground for support whereas the normal animal mainly walks on the toes. Similarly, total toe spread and intermediary toe spread values represented a reliable and sensitive method of measurement in evaluating functional improvement.
The quest for an ideal neurotrophic factor is still a distant dream. Towards this goal, the best functional result will occur when the axonal growth occurs in the proper direction, in the shortest time and with the best functional recovery. A variety of agents have been examined experimentally such as melanocortins, adrenocorticotrophic hormone (ACTH)) and α-melanocyte-stimulating hormone (α-MSH) (6,11). Recently, FK 506, widely used as an immunosuppressant, has been shown to enhance axonal regeneration in experimental animals and in vitro methods (7,12-14). However, the drug which has mostly replaced cyclosporin A as an immunosuppressant, was found to be severely, nephrotoxic and neurotoxic, both in animals and humans (15,16). Further, experiments with non-immunosuppressant analogues of FK 506, have shown equal potency in enhancing axonal regeneration (17-19).These studies, utilizing the rat sciatic nerve model have demonstrated that there is statistically significant growth both in the density of axonal fibers as well as faster rate of regeneration in the drug given animals when compared to the controls. However, given the numerous health risks associated with immunosuppression, whether the degree of improvement in a peripheral nerve injury is worth the risk could be answered only by additional studies. The most common approach to the problem of the nerve gap is nerve autografting, and although nerve grafting remains the gold standard in treating a significant nerve defect, the results are often suboptimal. The results of peripheral nerve grafting remain unpredictable and advances in the field of study have reached a technical impasse. The successful repair requires guidance of the regenerating axons towards the distal nerve segment, or these elements will grow in a disorganized fashion creating a neuroma. One of the more intensely studied alternatives is the use of a conduit to bridge the nerve defect. The ability of the conduit to guide axonal regeneration is based on the concept of neurotropism and neurotrophism, properties inherent to peripheral nerve tissue (20). Among these, the silastic, polyglycolic acid and other biodegradable tubes have shown statistically and clinically good results in enhancing nerve regeneration (21,22). Though veins grafts have been used widely (23), our experience with them was far from satisfactory due to its collapsibility. Recent studies showed that Matrigel, a biodegradable basement membrane matrix has the ability to enhance nerve regeneration but the exact mechanism remains unknown (24). Experiments utilizing this material impregnated with FK 506 as a local drug delivery system in a rat sciatic nerve crush model are presently underway in our lab.
Improvements in techniques and surgical methods discovered lasers and fibrin glue as alternative methods of nerve repair (25,26). Our experiments with different diameters of nylon ranging from 7-0 to 10-0 has not altered the functional recovery in a rat sciatic nerve transection model. We believe that the tension at the suture line is an important detrimental factor for poor functional recovery as emphasized by Terzis at el (27). Further, early repair and good vascular bed are some of the other factors which aid in promoting good functional result.
An ideal neurotrophic agent in enhancing nerve regeneration is far from a reality. Recent promise shown by FK 506 in experimental methods in rats falls short due to its toxicities when used systemically. However, local application of FK 506 as shown by Steiner et al (28) is an attractive proposition and will reduce the systemic effects of the drug. Future studies should focus on this technique and analogues of FK 506 which are non toxic, to explore further the potential benefit of these agents for the treatment of peripheral nerve injuries. Sciatic functional index is a sensitive method in evaluating functional recovery in animals. Our experiments with india ink was found to be simple, inexpensive and reasonably reliable technique in analyzing the foot prints of the animals.
Figure 1. Bar graph showing mean toe spread values. 1. The Interdigit measurements are larger when compared to the 2. Experimental animals measured at 18 days. Quantitative analysis of the foot prints was made directly from the original prints. The feet of the animals were dipped in india ink (black drawing ink) and made to walk on a plain sheet of paper.
Figure 2. Digital image of the foot print of the experimental animal enlarged from the imprints at 3 weeks. Note that the inter-digital distances are almost absent and the imprint of the fifth toe is absent indicating the deficit. In addition, the full print length (PL) of the foot is printed and the value is increased when compared to a normal animal (fig 3)
Figure 3. Digital image of a normal foot print of the rat taken directly from the india ink imprints. Note the widespread toes (TTS), (ITS) and short print length indicating that the normal animal walks mostly on the toes. Image to image size of figures 2 and 3 are not matched.
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