Ladder Test Research Articles

Behavioral and Histological Characterization of Unilateral Cervical Spinal Cord Contusion Injury in Rats

Most experimental studies of spinal cord injury (SCI) in rats damage the thoracic cord, with the consequent functional loss being due to interruption of long tracts connecting the caudal spinal cord to the rostral nervous system. Less work has been done evaluating injury to the cervical cord, even though it is the most common level of human SCI. In addition to the long tracts, the cervical spinal cord contains the sensory and motor neurons responsible for upper extremity function. The purpose of this study was to further develop a rat model of cervical spinal cord contusion injury using a modified NYU/MASCIS weight drop device. Mild (6.25 mm) and moderate (12.5 mm) C5 unilateral injuries were produced. Behavioral recovery was examined using a grooming test, a paw preference test, a walkway test (The Catwalk), and a horizontal ladder test. Histological outcome measures included sparing at the lesion epicenter, sparing throughout the extent of the lesion, quantification of myelin loss rostral and caudal to the lesion, and motor neuron counts. Compared to controls, animals receiving SCI exhibited injury severity-specific deficits in forelimb, locomotor, and hindlimb function persisting for 6-weeks post-SCI. Histological analysis revealed ipsilateral containment of the injury, and differentiation between groups on all measures except motor neuron counts. This model has many advantages: (1) minimal animal care requirements post-SCI, (2) within subject controls, (3) functional loss involves primarily the ipsilateral forelimb, and (4) it is a behavioral and histological model for both gray and white matter damage caused by contusive SCI.

Minocycline and intracerebral hemorrhage: influence of injury severity and delay to treatment

Intracerebral hemorrhage (ICH) is a devastating condition currently lacking a defined line of treatment. The inflammatory response that ensues following its onset is thought to contribute to secondary injury following ICH, making inflammation a potential therapeutic target. Minocycline (MC), a commonly used antibiotic that also has anti-inflammatory and anti-apoptotic properties, provides histological protection in several animal stroke models when given soon after injury. However, its ability to provide protection with more clinically relevant delays is unknown. The objective of this study was to examine the effects of MC on histopathological changes and long-term functional outcomes in a collagenase-induced ICH model in rats when drug administration was delayed 3 h following the onset of ICH. In accordance with other studies, MC suppressed microglial/macrophage activation in the peri-infarct region at 5 days based on B 4 isolectin histochemistry. However, no reduction in infarct volume was detected at 5 or 28 days post-ICH. Minocycline given for either 5 or 14 days also provided no functional benefit as assessed with a battery of sensory-motor tests (i.e., staircase, cylinder, ladder tests). These findings raise questions about the ability of MC to provide protection in ICH when delay to treatment is increased.

Considerations in the selection of behavioural tests to gauge recovery after intracerebral hemorrhagic stroke in rats

Cerebral ischemia and hemorrhage (stroke) are leading causes of death and disability. The goal of experimental cytoprotectants (neuroprotection) for stroke is to reduce cell death and thereby improve functional outcome. Rodents (rat, gerbil, mouse) are most commonly used to model ischemic and hemorrhagic insults. Despite the fact that behavioral recovery is the clinical endpoint of greater concern, many cytoprotection studies rely solely upon histopathological endpoints (e.g., infarct volume) to gauge treatment efficacy (DeBow et al., 2003; Can. J. Neurol. Sci). Furthermore, those studies that assess recovery often use only a neurological deficit score (NDS; e.g., paw placement, spontaneous rotation) and do not use more demanding / informative tests (e.g., skilled reaching). Successful translation of putative cytoprotectants from rodent models to human will depend upon how well treatments are initially assessed. Many functional tests are available in the rat and have been used in focal ischemia studies. Presently, we used a battery of functional tests, which are sensitive to sensory and motor system dysfunction, to gauge outcome after an intracerebral hemorrhagic stroke (ICH) largely targeting the striatum. Sixty adult male Sprague Dawley rats (N = 15 per group) were stereotaxically infused with 1 L of sterile saline containing 0 (SHAM), 0.06 (MILD lesion), 0.12 (MODERATE lesion), or 0.18 U (SEVERE lesion) of bacterial collagenase. Testing began one day following ICH and was repeated over 30 days. Tests included the horizontal ladder and beam walking tests, swimming, limb-use asymmetry (cylinder) test, a NDS, an adhesive tape removal test of sensory neglect, and the staircase and single pellet tests of skilled reaching. At present, the NDS, staircase, neglect and single-pellet tests have been analyzed. Overall, these tests revealed significant impairments (vs. SHAM) that improved over time (e.g., deficits on the NDS were greatest on days 1 and 2). However, despite significant differences in lesion size, we were often unable to statistically distinguish between MILD and MODERATE or MODERATE and SEVERE groups (e.g., neglect test). As for skilled reaching, the staircase test revealed significant contralateral forelimb impairments in the three ICH groups and was often able to distinguish among ICH groups. The SEVERE group also had significant ipsilateral impairments. Skilled reaching impairments were also found with the single-pellet test in the ICH groups. However, it was not possible to compare success among ICH groups because many rats in the MODERATE and SEVERE groups completely switched limb preference. Indeed, a detailed analysis of the entire reaching sequence with the contralateral limb was only possible in the SHAM and MILD groups. In conclusion, multiple behavioral tests are needed to comprehensively evaluate functional recovery after ICH. However, some tests (e.g., neglect test) are either not recommended for this model owing to the time required to test animals, variability, and the insensitivity to lesion size, or are of limited use (e.g., for small lesions). Finally, our results show that modest neuroprotection (e.g., reducing lesion size from SEVERE to MODERATE) may not consistently improve functional recovery, thereby necessitating the use of several tests.

Growth inhibition and cell cycle arrest effects of epigallocatechin gallate in the NBT-II bladder tumour cell line.

To examine the growth inhibition and cell cycle arrest effects of epigallocatechin gallate (EGCG), a major constituent of green tea polyphenols, on the NBT-II bladder tumour cell line. Growth inhibition and cell cycle arrest effects of EGCG were evaluated by the tetrazolium assay, flow cytometry and apoptotic DNA ladder tests. The cell cycle-related oncogene and protein expressions in NBT-II bladder tumour cells, when incubated with EGCG, were detected with reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. EGCG inhibited growth of the NBT-II bladder tumour cells in a dose- and time-dependent manner. Flow cytometry showed a G0/G1 arrest in cells when cultured with EGCG at doses of 10, 20 or 40 micro mol/L for 48 or 72 h. The apoptotic DNA ladder test showed that EGCG at 10 micro mol/L induced early apoptosis after 48 h of incubation. A down-regulation of cyclin D1 was detected by RT-PCR when the cells were incubated with EGCG (20 micro mol/L for 48 h. EGCG also down-regulated protein expression of cyclin D1, cyclin-dependent kinase 4/6 and phosphorylated retinoblastoma protein, in both a time- and dose-dependent manner, when detected by Western blot. EGCG had growth inhibition and cell-cycle arrest effects in NBT-II bladder tumour cells by down-regulating the cyclin D1, cyclin-dependent kinase 4/6 and retinoblastoma protein machinery for regulating cell-cycle progression.

Development of early motor learning and topical motor skills in a model of cerebellar degeneration

The development of motor learning and topical motor skills has been studied during the first month of life in neurodeficient Lurcher mutant mice derived from C3H strain. The examination of motor functions was conducted using four methods when animals were consecutively placed on: a horizontal wire, a rotating cylinder, a bridge and slanting ladder (on the rotating cylinder and slanting ladder in three different starting positions). A total of 150 animals were used, half being heterozygous mutants (+/Lc), the remaining consisting of their healthy littermates, homozygous wild-type mice (+/+). Some animals of both types were confronted with the tasks in four testing days without practice while the others were trained in all of these methods one time daily at the age of 3, 6, 9, 12, 15, 20, 25 and 30 days. The results of the research are expressed as a percentage of reaching criteria in the trained and untrained mice of both types (+/Lc, +/+), respectively on days 15, 20, 25, and 30. Because of their motor handicap, Lurcher mutants showed significantly worse results than normal mice in almost all of the tests. The most interesting observations of the study were obtained on the rotating cylinder and the slanting ladder tests where mainly untrained mutants were unable to cope promptly with the different starting position and failed. However, in 30-day-old Lurchers the effect of learning was relatively better in some tests when compared with the wild-type of mice. Experiments showed that, in spite of progressive cerebellar degeneration, the ability of motor learning in Lurcher mutants survived to a certain degree. The worsening of results after frequent training in both trained Lurchers and normal mice aged 20 days as compared with untrained ones is discussed within the context of the “overtraining reversal effect” and other possible hypotheses.

Design and characterization of a high-precision resistor ladder test structure

A new subsite stepped multiresistor test structure is introduced. This test structure is used for studying and improving small resistance mismatch patterns in resistor ladders for high-resolution analog-to-digital converter applications. By utilizing wafer prober subsite movements and contact pad cross connections in the test structures, in combination with a Kelvin measurement method and dedicated statistical data evaluation technique, this approach enables identification of very small (<0.05%) systematic resistance mismatch patterns in realistic high- precision resistor ladder implementation. The most disturbing mismatch pattern was found to be caused by mechanical stress from the resistor ladder head layout, while others are attributed to decananometer scale reticle writing artefacts.

Resistance of SKW6 cell to apoptosis induction with anti-Fas antibody upon transduction of a reverse fragment to a cDNA encoding human 6A8 α-mannosidase

The effect of transduction with a reverse fragment to a cDNA encoding human 6A8 alpha-mannosidase on apoptosis induction of human B cell line SKW6 by anti-Fas antibody was tested. Apoptosis-inducer of anti-Fas monoclonal antibody was used to induce apoptosis in SKW6 cells. Giemsa's staining, Annexin-V-FLUOS staining and DNA ladder test were used to determine the events of apoptosis. Indirect immunofluorescent staining with anti-Fas antibody was performed to detect the surface Fas expression. In a time-course test of 12, 24 and 36 h for apoptosis induction by anti-Fas antibody, DNA ladder was observed in the wild-type SKW6 cells in a time-dependent fashion. Mock transduction had no effect on DNA ladder production. However, no DNA ladder was detected in the rAAV-antisense 6A8 cDNA-transduced SKW6. Results from Annexin-V-FLUOS staining on anti-Fas antibody-treated cells revealed that the staining-positive rate in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells was decreased in comparison to that in the wild-type and the mock-transduced cells. Giemsa's staining observation showed that the number of dying (with apoptotic bodies) and dead cells was reduced in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells in comparison with that in the wild-type and the mock-transduced cells upon anti-Fas antibody induction. The transduction did not affect the expression of Fas molecular on cell surface. 100% cells in all the groups showed Fas expression. The SKW6 cells became resistant to apoptosis induction by anti-Fas antibody upon transduction with a reverse fragment to a cDNA encoding human 6A8 alpha-mannosidase. The transduction did not affect the expression of Fas molecule on cells.

Microtrasplante de células mesencefálicas fetales en estriado y substantia nigra pars reticulata simultáneamente de ratas hemiparkinsonianas. Estudio conductual

Microtransplantation of fetal dopaminergic cells has been used over the past ten years with good results in models of Parkinson's disease. To evaluate the effect of microtransplantation of fetal dopaminergic cells 'seeded' in the substantia nigra pars reticulata (SNpr) and striate (St) simultaneously. The animals received a transplant or microtransplant of cells into the St and SNpr ipsilateral to the lesion in the substantia nigra pars compacta or to both regions. Depending on the site and technique used the following experimental groups were considered: I. Macrotransplantation to the St (n = 20); II. Microtransplant to the St (n = 20); III. Microtransplant to St + SNpr (n = 20); IV. Microtransplant to St + SNpr (n = 20); V. Macrotransplantation to SNpr (n = 20); VI. Microtransplantation to SNpr (n = 20); and VII. Control (lesion only) (n = 20). The rotations induced by D-amphetamine (5 mg/kg i.p.) and by apomorphine were studied 1, 2, 3 and 6 months and 3 and 6 months respectively after transplantation. Three months after transplantation we studied the motor asymmetry shown by the animals by means of the ladder test. The rotations were reduced in the groups with intrastriate transplantation. Comparison between the surgical techniques showed nonsignificant differences between them. The ladder test showed significant differences in use of the limbs in all experimental groups. Use of the left limb was significantly reduced in all groups. Modification of the rotations seems more sensitive to the site of transplant than to the technique used. It seems that the skills studied using the ladder test are not altered by the microtransplant technique.

Trasplante simultáneo de células mesencefálicas fetales en striatum y globus pallidus de ratas con lesión por 6-hidroxidopamina

Studies of neural transplants in experimental models of Parkinson's disease have concentrated their attention on ectopic transplants of foetal mesencephalic cells to denervated striatum. However, the external globus pallidus has recently been shown to play an important part in the physiopathology of this disease. Bearing in mind the importance of loss of extra-striatal dopamine in the genesis of the clinical signs found in parkinsonism, the objective of this study was to evaluate the effect of foetal mesencephalic transplantation to the globus pallidus of hemiparkinsonian rats. Following conventional transplantation methodolgy, suspensions of cells from the ventral mesencephalum of rat embryos (E-14) were implanted. The tissue was grafted into the striatum, pallidum-striatum and pallidum areas of rats with unilateral lesions of the striatonigral bundle. One, two, three and six months after transplantation, the rotatory activity induced by D-amphetamine was evaluated. The rotatory behaviour induced by apomorphine was evaluated at three months. Motor ability of the front legs was evaluated in all experimental groups three months after transplantation using the 'ladder test'. In the experimental groups in which a transplant was made to the globus pallidus there was a significant reduction (p < 0.01) in rotatory activity induced by D-amphetamine and by apomorphine as compared with the non-transplanted groups. Transplants of foetal dopaminergic cells survive in the globus pallidus of hemiparkinsonian rats and can improve the rotational activity induced by dopaminergic agonists.

Evaluación conductual del modelo de lesión unilateral en ratas con 6-hidroxidopamina. Correlación entre las rotaciones inducidas por D anfetamina, apomorfina y la prueba de habilidades manuales

Evaluation of rotatory activity induced by dopaminergic agonists is the most widely used test of conduct for the measurement of dopaminergic depletion of a unilateral lesion of the striatonigral pathway caused by 6-hydroxydopamine (6-OHDA) in rats, since it is quantitatively related to the extension of the dopaminergic denervation. The objective of this study was to evaluate, from different angles, the changes in conduct seen in the model of unilateral lesion with 6-OHDA and to establish correlation with the rotation induced by D-amphetamine and by apomorphine and the ladder test. Male Wistar rats were used. Lesions were produced in the SNpc by stereotactic injection of 6-OHDA into the right hemisphere and the effectiveness of the lesions was studied using the rotary conduct induced by D-amphetamine and apomorphine. The motor ability of the front legs was measured by the ladder test, carried out under standard and forced conditions. All the animals with lesions had difficulty in reaching food with both legs, although the most pronounced deficit was in the leg contralateral to the lesion. The ladder test correlated better with rotatory activity induced by apomorphine than by D-amphetamine. The animals with most dopamine loss showed most deficient use of their front legs.

Transforming growth factor-beta1 enhances the lethal effects of DNA-damaging agents in a human lung-cancer cell line.

In tissue culture conditions, exogeneous active transforming growth factor-beta1 (TGF-beta1) enhances the lethal effect of DNA-damaging agents (UV-C, gamma rays, cisplatin, methotrexate and 5-fluorouracil) toward human A549 cells and mink Mv1Lu cells, as detected by the loss of their capacity to give rise to colonies; both these cell lines harbor a wild-type p53, as determined by immunoprecipitation. Contrastingly, the sole effect of the cytokine used alone is to inhibit reversibly the multiplication of the same cells without further impairing, once withdrawn from their environment, their capacity to divide and give rise to colonies. The lethal synergy between TGF-beta1 and UV-C was studied on mink and human cell lines, and the biomodulation by TGF-beta1 of cell killing by cisplatin, gamma rays, 5-fluorouracil or methotrexate was tested only on human cells. As investigated with UV-C-irradiated human A549 cells, TGF-beta1 appears to enhance apoptosis rather than to disturb the repair of DNA photolesions (mainly pyrimidine dimers) by the nucleotidic excision repair pathway according to results of nucleosomal ladder and comet tests. Our data raise the possibility that, in vivo, TGF-beta1 might affect the curative and/or undesirable secondary side effects of cancer therapy.

Step stress tests: Monte Carlo simulation and evaluation

In this technical note the historical development of step stress tests is briefly treated. Monte Carlo simulation of such tests has been applied to the case of simple tests (one ladder of stresses), wherein an Arrhenius reaction rate model is considered and semiconductor cumulative failure distribution vs. time is assumed to be lognormal at constant stress. Calculated results for activation energy are obtained by means of a computer program. It is concluded that the smaller the step height, the better the results and also that simple tests have an advantage over two ladder tests because they don't require a minimum step height nor previous knowledge of the activation energy.

PREDICTION OF MOTOR SKILL ATTAINMENT FROM EARLY LEARNING.

Learning of a whole-body motor task (ladder test) progresses by a two-component exponential curve. The fast component lasts only 5 trials, while the slow component, established within the first 10 trials, controls the mean trend for a 60-trial learning period distributed over 6 days. Warm-up decrement is noticeable on the last 3 days. Individual differences in initial skill have relatively little relation to performance attainment after the first 3 days. Reasonably good multiple-correlational prediction of individual attainment in this task after n days of practice requires using initial skill estimates combined with learning scores that cover at least the first n/2 days of practice.