Correlation between different methods of gait functional evaluation in rats with ischiatic nerve crushing injuries

Abstracts

Foi realizado um estudo da correlação entre diferentes métodos de avaliação funcional do nervo ciático de ratos após lesão por esmagamento. Foram empregados no estudo 25 ratos da linhagem Wistar, de peso médio em torno de 250 g, submetidos a esmagamento controlado do nervo e avaliados por dois métodos convencionais para obtenção do índice funcional do ciático (IFC), baseados respectivamente na medida manual e computadorizada de parâmetros das impressões das pegadas dos animais, e por um novo método desenvolvido pelos autores, baseado na filmagem das pegadas e medida dos mesmos parâmetros, no 1º, 7º, 14º e 21º dias de pós-operatório, sendo feita a comparação com os valores obtidos no pré-operatório e entre os diferentes métodos nos vários períodos. Os resultados mostraram que o método da medida dos parâmetros pela filmagem das pegadas permite melhor visualização da pata acometida pela lesão do nervo e que este método se correlaciona com positivamente com os convencionais, já a partir da primeira semana de pós-operatório, desde que seja empregada a mesma fórmula para o cálculo do IFC.

Avaliação; Nervo ciático; Regeneração

ORIGINAL ARTICLE

Correlation between different methods of gait functional evaluation in rats with ischiatic nerve crushing injuries

Andréa Licre Pessina Gasparini^I; Cláudio Henrique Barbieri^II; Nilton Mazzer^III

^IPhysical Therapist, Ph.D. Student of the Post-Graduation Program in Orthopaedics, Traumatologyand Rehabilitation. Department of Biomechanics, Medicine and Locomotive Apparatus Rehabilitation – FMRP – USP

^IIChairman of the Department of Biomechanics, Medicine and Locomotive Apparatus Rehabilitation – FMRP – USP

^IIIAssociate Professor, Department of Biomechanics, Medicine and Locomotive Apparatus Rehabilitation – FMRP – USP

^{Correspondences to}

SUMMARY

A study of the correlation between different methods of functional evaluation of the sciatic nerve of rats after a crushing injury was carried out. Twenty five Wistar rats weighing 250 g in average were submitted to a controlled crushing injury of the nerve and evaluated by two conventional methods for obtaining the sciatic functional index (SFI), based on manual and computer-based measurement of parameters on animals' footprints, and by a new method developed by the authors, based on walking track movie recording and the measurement of the same parameters, on the 1^st, 7^th, 14^th and 21^st postoperative days, a comparison being established with the values obtained preoperatively and between the different methods in all periods. The results showed that the method of recording the walking track allows for a better visualization of the injured paw and that this method positively correlates with the conventional methods, as early as the first postoperative week on, provided that the same SFI formula is used.

Keywords: Evaluation; Sciatic nerve; Regeneration

INTRODUCTION

Functional evaluation of peripheral nerve injury in animals is a challenging experimental factor. After nervous injury, morphological and functional recovery is seldom full and perfect, despite of the use of modern and sophisticated reconstruction techniques^(1,2). In experimental environments, injuries recovery is studied basically by histology and morphometry techniques^(3,4). Although these processes are useful, it is important to follow-up an injury functional recovery. Experimental studies represent an important research source for understanding injuries and peripheral nerves regeneration^(5,6). Expert literature is abundant in experimental studies focusing injury^(7,8). The use of therapeutic resources is a highlight on regeneration^(9,10). A significant focus is driven to the study of functional recovery evaluation, which is always difficult in animals, for understandable reasons^(11,12,13).

De Medinaceli et al.⁽¹⁴⁾, developed a quantitative, reproducible method of the functional status of rats' sciatic nerves by analyzing characteristic data from animals' footprints. This method was modified in 1984, with the addition of information technology resources, making it more practical, faster and user-friendly, keeping the measurement of the so-called Sciatic Functional Index by means of the formula:

Other researchers modified the existent methods by mathematic formulas, excluding parameters of significant statistical variance^(15,16).

Bain et al.⁽⁵⁾ conducted an experimental study in rats intending to develop and test independent and more reliable equations for functional evaluation of sciatic, tibial and peroneal nerves regeneration, by using the following mathematic formulas:

Despite those modifications, limiting factors for using the method still existed, making it difficult to apply, such as, for example, the need of hardware with specific software for addressing walking track, and even how to obtain paw prints.

Different techniques have been developed for properly capturing an animal's footprint. Real-time image capturing by means of videotape recording, was an advance in the experimental field of functional evaluation of gait in rats with peripheral nervous injuries^(3,13). Visualization, recorded with no footprint failures, enables an easier marking of required measures for obtaining the Sciatic Functional Index (SFI).

Despite of the available literature addressing the use of different methods of functional evaluation, no studies are found based on a correlation between such methods. Thus, we intended to submit animals with sciatic nerve crushing, continuous-load injuries to known functional evaluation methodologies and to adapt videotaping methodology to a software assessing gait function since the first postoperative day, presenting an easier analysis of the footprints, in addition to establish a correlation between those methodologies.

MATERIALS AND METHODS

For this experiment, 25 male Wistar rats weighting 250 g in average (range: 200 – 300 grams), kept in collective cages containing three animals each, in a temperature-controlled environment, with free access to food (specific standard Laboratory animal food) and water, up to the 21st postoperative day, deadline for experiment completion.

For surgical procedure, the animals were anesthetized by using a blend of 5% Ketamine and 2% Xylazine at 1:4 ratio, applied intraperitoneally at a 0.10 ml/100 g body weight dosage. Subsequently, the animals were weighted. Each animal was identified, and the right lower limb prepared for surgical procedure as usual, including trichotomy, antiseptic solution and site protection with surgical drapes.

The animals were positioned at ventral decubitus, with paws tied together in abduction on a proper support, and the sciatic nerve was addressed through a longitudinal straight lateral skin incision, about 3-cm long. The incision extended from femoral major tubercle to lateral femoral condyle, followed by blunt dissection between ischiotibial and major gluteus muscles, exposing the nerve from its emergence to its distal trifurcation. A domestic-use sterilized cotton wire loop was placed around the nerve for easier handling purposes, and the animals were then transferred to a dead-weight device, especially made for producing a crushing injury with a load of 15,000 g for 10 minutes, involving a 5-mm long segment, just proximal to nerve trifurcation⁽¹⁶⁾. (Figures 1 and 2).

After the load was applied, it was removed and the nerve was mobilized with the aid of the cotton loop. Surgical wound was closed with tissue suture by planes. The area was washed with 20% iodine alcohol antiseptic solution, and the animals received a single antibiotic dose for infection prophylaxis, being placed back into their original cages.

Each animal's back paws prints were recorded by using three different methods, at preoperative period, and on the 1st, 7th, 14th, and 21st postoperative days.

For assessing footprints and for calculating the Sciatic Functional Index, according to the method by De Medinaceli et al.⁽¹⁴⁾ , the animals were submitted to walk on a 43-cm long vs. 8.7 cm wide vs. 5.5-cm high wooden track, with a dark small shelter at the end. On the walking track, paper strips were applied with the same measurements, previously prepared (bromophenol blue solution (Sigma^®) in acetone, allowed to dry and stored in plastic containers in a dry environment). The animals had their paws wet by a water detergent domestic solution, which minimizes footprints blurring. (Figure 3).

Footprints images on paper strips were digitalized on a scanner and stored in a computer with a developed built-in graphical analysis software ^* routinely used in Laboratory^(6,9,17). That software, built on Windows^® platform, allows for capturing, storing and assessing footprints images, providing Sciatic Functional Index calculation by the methods by De Medinaceli et al.⁽¹⁴⁾ Carlton and Goldberg⁽¹⁵⁾ and Bain et al.⁽⁵⁾.

Those paper strips containing animals' footprints were manually assessed. Animals' paws measurements were highlighted and, with the aid of a school rule, with 1-cm increments, the values were calculated and stored. Subsequently, these data were included on a worksheet built on Excel application (Windows^®), providing Sciatic Functional Index calculation based on the mathematic formula by De Medinaceli.

For the analysis of videotaped walking track prints and calculation of the Sciatic Functional Index, according to the method by Bain et al⁽⁵⁾, the animals had to walk on a transparent acrylic track of similar size as the wooden track used with the method described above, but closed at its upper portion in order to prevent rats from escaping. Similarly, the animal positioned on the walking track had to walk all the way through towards a dark small shelter. The walking track was built with lighting on both sides with 120 W fluorescent lamps and gait recording was provided by using a digital video camera SONY DCR – DVD 203, positioned beneath the track. (Figure 4).

The footprints images obtained from the videotape were addressed by computer by means of the Image J software, and edited for transforming pixels into millimeters, in order to allow for the calculation of Sciatic Functional Index modified by Bain et al.⁽⁵⁾ (Figures 5 and 6).

The Sciatic Functional Index, which corresponds to gait functional analysis, was calculated on footprints obtained from the methods described above, using the formula modified by Bain et al.⁽⁵⁾ as follows:

Where:

N: normal, or non-operated; E: experimental, or operated; PL: print length; TS: total toe spread, or distance between first to fifth toe; IT: intermediate toes spread, or the distance between the second and fourth toes.

For statistical treatment of data, we used the Pearson's correlation coefficient (r), which quantifies the degree in which two variables (x and y) are correlated, provided the correlation is linear. This coefficient has no measurement unit, and ranges from -1 to 1. If r=0, no linear correlation exists between x and y, and the variables are not correlated. In order to assure results, we used the Spearman's rank correlation coefficient, r_s.

RESULTS

In the three respective methods, at first, the animals did not apply load on the injured paw, thus dragging it; keeping toes at full adduction, as a result of a serious sciatic nerve dysfunction. Over the experiment, the animals gradually recovered their ability to apply load on the injured paw and to open outer toes first, followed by intermediate toes.

Correlation between different gait functional evaluation methods manual postoperative SFI and computed postoperative SFI

A total of 125 walking tracks, each one with at least three footprints were measured by manual and computed methods by means of white paper sheets prepared with bromophenol. The same number of tracks was used, as image pictures, for assessing the videotape.

In the correlation established between Sciatic Functional Index (SFI) at manual and computed postoperative analysis, the Pearson's correlation coefficient was r = 0.54, thus determining a positive correlation between both, because as the sciatic functional index increases on the computed method, so increases the manual sciatic functional index. Upon data distribution, the Spearman's rank correlation coefficient was also applied. The p-value result of r (0.0108) was lower than the significance level (p<0.05), thus ruling out the H₀ hypothesis (no correlation), assuring correlation between studied methods (Figure 7).

Manual postoperative SFI and postoperative SFI with videotaping

Data concerned to the correlation established between the Sciatic Functional Index (SFI) at manual postoperative analysis and at the postoperative analysis with videotaping result in a Pearson's correlation coefficient of r = 0.32, determining a positive correlation between both, because as the sciatic functional index increases on the videotape method, so increases the manual sciatic functional index. Attention should be given to an atypical point on the graph that impacts values distribution, which attributes a curve shift towards that point. In this case, applying the Spearman's rank correlation coefficient was crucial, revealing a value of r_{s =} 0.40. The statistical hypotheses test considering a p-value < 0.05 (fixed significance level) for checking the reliability of values recorded for Pearson and Spearman, assured correlation (p-value of r = 0.0558; p-value of r_s = 0.0245). (Figure 8).

Computed postoperative SFI and postoperative SFI with videotaping

The analysis of the correlation established between Sciatic Functional Index (SFI) on computed postoperative and postoperative SFI with videotaping called our attention, because the Pearson's correlation coefficient was r ₌ 0.36 determining a positive correlation between both. The Spearman's rank correlation revealed a value of r_{s =} 0,29. These data alone characterize a positive correlation between both, that is, a linear distribution of the values obtained for Sciatic Functional Index. However, when values are distributed on the graph, there is no linearity, but a heterogeneous distribution of data. Thus, the reliability of r and r_s values with the statistical hypotheses test considering a p-value < 0,05 was confirmed, where the absence of correlation between methods was detected (p-value r= 0.1310; p-value r_s = 0.1877). (Figure 9).

DISCUSSION

Peripheral nerves injuries are always a reason for concerns in terms of functional recovery, because it is not always full^(1,2), sometimes leaving permanent sequels, which has been encouraging a large number of scientific investigations about the uncountable variables involved on nervous regeneration process. A large portion of those investigations are experimental by nature, conducted on animals, usually submitted to invasive evaluation methods, such as electroneuromyography, which does not anticipate information about an animal's functional status, or terminal methods, requiring animals' sacrifice and removal of the nerve for histomorphometric studies^(4,11). On the other hand, functional evaluation in animals is always an issue, for understandable reasons, being very difficult, for example, to quantify sensitiveness, or even motricity.

Gutmann and Gutmann⁽¹⁸⁾ noticed that, in rabbits, the ability to spread back paw's toes depended on peroneal nerve's functional integrity. This was the starting point for the development of a new functional evaluation method in rats submitted to sciatic nerve crushing injury and analysis of back paws prints, named Sciatic Functional Index, or SFI ⁽¹⁴⁾, which was subsequently modified^(5,15).

Since then, functional evaluation by SFI was shown to be a warranted reproducibility method with a strong correlation with the degree of morphologic regeneration⁽⁶⁾. SFI is, in fact, a negative indicator of nerve dysfunction degree, ranging from–100 to zero, -100 meaning full dysfunction, and zero, normal function. This method is based on fixed parameters measurable from footprints and their input on simple mathematic formulas, though subjected to errors.

Varejão et al.⁽¹³⁾, suggested a new evaluation method by direct inspection by means of videotaping rats' footprints, advocating that this method would assure good evaluation, even in early weeks after injury, when difficulties in obtaining data are reported. However, in another experimental study, based on the measurement of an angle of the animal's paw (TOA – toe-out-angle), using the calcaneus and the third and fifth toes as reference points, and assessed by means of computed analyses⁽²⁾, that group concluded that this method has a good correlation with the SFI by De Medinaceli⁽¹⁴⁾. However, the method described by Varejão et al.⁽¹³⁾ is more difficult to apply and largely depends on investigator's subjectivity.

Despite the existence of studies in which the SFI methods described by De Medinaceli et al.⁽¹⁴⁾ and Varejão et al.⁽¹³⁾ have been used, it seemed to us that the functional differences between both were not clear enough, which motivated us to develop the present investigation, not only for comparing both methods, but also to correlate them, applying the methodology described by the previous and modified by Bain et al.⁽⁵⁾ to the latter, thus creating a third new method (walking track videotaping plus parameters measurement), and then comparing to conventional methods that originated it, performed manually and with the aid of a computer. The controlled sciatic nerve crushing injury model in rats was regarded as optimal for this investigation, because this kind of injury can be produced easier than section followed by microsurgical repair. Additionally, in a crushing injury, neural structure is partially preserved, favoring nerve rege-neration within a shorter term. Crushing was provided for 10 minutes, with a 15,000-g load, in order to achieve a severe injury, especially on thicker fibers, particularly assuring motor dysfunction. Rats' back paws images were printed on paper soaked with bromophenol blue, and animals' gait was videotaped and subsequently digitalized and evaluated by the computer imaging software Image J, which allows for an easy visualization of the footprints and for the application of the method by De Medinaceli et al.⁽¹⁴⁾, as early as the first weeks after injury. Collected data were applied to the formula described by Bain et al^.(5), which was modified from the one by De Medinaceli et al⁽¹⁹⁾ and excluding the TOF (distance to-other-foot) parameter, thus eliminating the risk of results variations imposed by gait speed and step distance.

Our results demonstrate correlation between the SFI formulas by De Medinaceli et al.⁽¹⁴⁾ and by Bain et al.⁽⁵⁾ , in spite of the modification introduced in the latter (TOF parameter exclusion). From the analysis in which the correlation coefficient seeks to establish between manual SFI data and videotape SFI data, the distribution profile of achieved data shows to be different, with values being generated at significance threshold point, generating a distribution with positions tending to disorganization, almost without linearity, , but the statistical test assures positive linearity, establishing a correlation between each other. We thought, thus, that the linearity unbalance factor was the use of the original De Medinaceli et al.⁽¹⁴⁾ formula correlated to the modified formula by Bain et al. ^(5). An exception made to literature considering the TOF measurement as causative of potential variables in finding the SFI ^(5,15). In the attempt to correlate computed SFI values and videotape SFI values, a disorganized distribution of data is found, which is not supported by statistical test, thus characterizing lack of correlation between data.

CONCLUSION

Functional evaluation methods described in this experimental study correspond to tools that assure regeneration and functional recovery follow-up, where the hypothesis of correlation between methods is real for conditions in which the same mathematic calculus is used, regardless of being manual or computed, with identification of the same measurement parameters. In cases where a combination of mathematic formulas exist targeting SFI, and the parameters measured are different from each other, there is no correlation between methods, reinforcing the use of individual formulas for separated experiments. Upon the need of accurate records, even in the first weeks, we conclude that the functional evaluation method with videotaping is the best one for regeneration follow-up, with immediate footprint data, mitigating difficulties faced with the traditional method by De Medinaceli.

ACNOWLEDGEMENTS

The authors acknowledge Professor José Batista Volpon and the FMRP - USP Bioengineering Laboratory staff, Professor Amilton Antunes Barreira, from the Department of Neurology and Psychiatry for being so kind to approve our use of the laboratory equipment, and Mr. Renato Meirelles e Silva and Mrs. Maria Cristina Lopes Schiavoni for their technical assistance. We also acknowledge Rodrigo Okubo, student of the Physical Therapy Course at USP Ribeirão Preto, for contributing with his technical knowledge on image recording and edition.

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Received in: 16/010/06

approved in: 02/20/07

Study conducted at the Department of Biomechanics, Medicine and Locomotive Apparatus Rehabilitation, Ribeirão Preto, University of São Paulo.

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Publication Dates

• Publication in this collection
  31 Jan 2008
• Date of issue
  2007

History

• Received
  16 Oct 2006
• Accepted
  20 Feb 2007