Habilitation Improves Mouse Gait Development Following Neonatal Brain Injury

ABSTRACT

Objectives: Neonatal brain injury during gait development disrupts neural circuits and causes permanent gait dysfunction. Rehabilitation as an intervention to improve impaired gait function has been used in adults as a treatment for stroke and spinal cord injury. However, although neonates have greater neuroplasticity and regenerative capacity than adults, normal gait development and the effects of habilitation on gait function following neonatal brain injury are largely unknown.

Methods: In this study, we generated cryogenic injury in mice at postnatal day 2 and subsequently performed habilitative training to promote autonomous limb movement for 4 weeks. We also quantitatively analyzed the gait acquisition process in developing mice using the Catwalk XT system.

Results: Using quantitative gait analyses, we showed that during normal gait development in mice, stance phase function matures later than swing phase function. We also demonstrated that habilitation in which active limb movements were enhanced by suspending mice with a rubber band with no floor grounding promotes motor learning, including gait function, in mice with impaired acquisition of gait function resulting from neonatal brain injury.

Conclusions: Our findings provide a basis for research on gait development in mice and suggest new habilitation strategies for patients with impaired gait development caused by perinatal brain diseases such as hypoxic–ischemic encephalopathy and periventricular leukomalacia.

INTRODUCTION

Neonatal brain injury in humans occurs at a rate of one in a thousand.¹⁾ Typical perinatal neurological diseases, such as hypoxic–ischemic encephalopathy and periventricular leukomalacia, are important social problems because they are causative diseases of mental retardation and gait development disorders. These neonatal lesions in the central nervous system (CNS) are irreversible, and no effective treatment has been established.

Rehabilitation can facilitate the acquisition of physical functions that have been impaired. In adults, gait function impaired by stroke or spinal cord injury can be improved by various rehabilitation methods, including the use of robots,²⁾ gait training in a partially or completely unloaded state,³⁾ and the use of a Visual Display Terminal.⁴⁾ However, the facilitation of physical function acquisition following neonatal brain injury is more complicated because it does not involve regaining impaired functions but acquiring new physical functions with impaired brain. This process to assist the development of motor skills that have not yet been acquired is called “habilitation.” In neonates, early habilitation of children born with periventricular brain injury reportedly promotes acquisition of motor skills,⁵⁾ but it is not clear whether early habilitation promotes gait function. Neonates have higher neuroplasticity^6,7) and regenerative capacity than adults,^8,9) and, therefore, supporting the gait acquisition process through habilitation may be a promising treatment for perinatal neurological diseases. However, the details of such a habilitation process are still unclear.

Because of their amenity to various experimental interventions and genetic manipulations, mice are useful laboratory animals for studying impaired gait behaviors caused by CNS injuries and their treatment. The rotarod test, foot fault test, and beam walking test have been used to detect injury-induced gait abnormalities in adult mice.^10,11,12) Recently, comprehensive gait parameter evaluations in adult mice using automatic gait analyzers such as Catwalk and DigiGait have also been reported.^13,14,15,16) However, the process of acquisition of normal gait and the effects of injury and habilitation have not been well studied in neonatal mice.

In this study, using Catwalk, we quantitatively analyzed the gait acquisition process in developing mice and clarified the mechanism of the normal gait acquisition process. Furthermore, we found that habilitation methods referred to as “air-walk habilitation” promote motor learning recovery of gait function after impaired acquisition of motor function caused by neonatal brain injury.

MATERIALS AND METHODS

Animals

Institute of Cancer Research mice were purchased from Japan SLC. All the animals used in this study were maintained with a mother mouse during weaning or in groups of seven mice per cage after weaning and on a 12-h light/dark cycle with ad libitum access to food and water. The experiments involving live animals were all performed in accordance with the guidelines and regulations of Nagoya City University (Approval No. 21–028).

Neonatal Brain Injury and Habilitation

Postnatal day 2 (P2) pups were subjected to cryogenic injury as described previously.^17,18) Briefly, the pups were anesthetized using isoflurane, and the skull was exposed through a scalp incision. A 1.5-mm-diameter metal probe cooled by liquid nitrogen was stereotaxically placed on the right skull (0.5 mm anterior and 1.2 mm lateral to the bregma), for 30 s. The scalp was immediately sutured with an adhesive, and the mice were returned to the home cage.

Air-walk habilitation training was performed by suspending pups with rubber bands, as shown in Fig. 1B; this condition promotes autonomous movement of limbs without ground loading. When the mice stopped moving during suspension, the limbs were stimulated by finger touch to promote swinging. Air-walk habilitation was performed for 20 min/day from the day after cryogenic injury (P3) to P30. Seven mice per group (i.e., control, injury, and habilitation groups) were used in this study.

Fig. 1.

Effect of habilitation following neonatal brain injury on body weight change and injury volume in mice. (A) Experimental scheme. Cryogenic injury was performed at postnatal day 2 (P2) and body weight was measured at 1, 2, 3, and 4 weeks post-injury (wpi) in control (no injury), injury (injury and no habilitation), and habilitation (injury and habilitation) groups. The injured volume was measured at 0.5 wpi (4 days post-injury) and 4 wpi. (B) Schematic illustration of habilitation. Mice were suspended using rubber bands, which allowed active locomotion of their limbs. (C) Body weight change at 1–4 wpi in control, injury, and habilitation groups. Data shown are mean ± SEM. (D) Representative images of coronal sections of the cerebral cortex at 0.5 (D, left) and 4 (D, right) wpi in control, injury, and habilitation groups stained for GFAP (D, red) and Iba1 (D, green). Nuclei were stained with Hoechst 33342 (white). Scale bar, 1 mm. (E) Injured area at 0.5 and 4 wpi in injury and habilitation groups. Data shown are mean ± SEM.

Gait Behavior Analyses

The foot-fault test was performed as reported previously.¹⁸⁾ Briefly, mice were placed on an elevated wire hexagonal grid with 40-mm-wide openings and were allowed to roam freely. A misstep was recorded as a foot fault if the mouse slipped or fell with one of its limbs dropping into an opening in the grid. The number of foot faults was counted for 5 min for each limb, and then the ratio of the number left forelimb and left hindlimb faults to the total number of faults for all four limbs was calculated as a percentage. The test was performed twice, and the values were averaged.

Quantitative gait analysis was performed, as reported previously,¹⁸⁾ using a Noldus CatWalk XT system (Noldus Information Technology, Wageningen, Netherlands). Briefly, the mice were allowed to walk across a glass walkway illuminated with a green light in a dark environment at 23 ± 1°C. The contact point of each paw on the glass was illuminated and was recorded with a high-speed video camera. To detect the contact intensity of each paw during gait development of postnatal mice (2–4 weeks old), the criteria used for the camera, contact intensity, and cutoff for gait duration were as follows: camera gain (dB), 20.00; green intensity threshold, 0.10; cutoff for gait duration of each animal, 0.50–5.00 s. At least three successful sustained walk recordings for each mouse were used for each analysis, and the average of the runs was reported. Details of the parameters used in the Catwalk analysis were as follows¹⁸⁾: body speed, i.e., the distance that the mouse’s body traveled from one initial contact of the paw to the next, divided by the time to travel that distance; swing speed, i.e., the speed of a paw during swing phase; the mean (max contact mean intensity) and maximum (max contact max intensity) pressures at maximum ground contact; and the print area, i.e., the total floor area of a paw that comes into contact with the plate during the stance phase.

Immunohistochemistry and Image Acquisition

Immunohistochemistry was performed as described previously.¹⁹⁾ Briefly, the brain was fixed by transcardiac perfusion with 4% paraformaldehyde in 0.1 M in phosphate buffer and postfixed overnight in the same fixative. Floating coronal sections (60 µm thick) were prepared using a vibratome (VT-1200S, Leica, Nussloch, Germany) and then incubated for 30 min at room temperature in blocking solution (10% normal donkey serum and 0.2% Triton X-100 in phosphate-buffered saline). The sections were then incubated overnight at 4°C with mouse anti-GFAP (1:1000, Sigma, St. Louis, MO) and goat anti-Iba1 (1:2000, Abcam, Cambridge, UK) antibodies, and then for 2 h at room temperature with Alexa Fluor-conjugated secondary antibodies (1:1000, Invitrogen, San Diego, CA) in blocking solution. Nuclei were stained with Hoechst 33342 (Sigma).

Images of coronal cortical sections were acquired by scanning at 2-µm intervals using an LSM 700 confocal laser-scanning microscope (Carl Zeiss, Oberkochen, Germany) with a 20× objective lens. The lesion area was defined as the inner region of a GFAP+ glial scar and was measured using ZEN software (Carl Zeiss).

Statistics

All statistical analyses were performed using SPSS (IBM, Armonk, NY). Sample sizes were chosen based on those used in previous studies. The normal distribution and variance between groups were statistically analyzed. Comparisons among multiple groups were analyzed by one-way analysis of variance followed by a post-hoc Tukey honestly significant difference test. The data are presented as the mean ± SEM and P-values less than 0.05 were considered to be statistically significant.

RESULTS

Habilitation Promotes Motor Learning of Gait Function in Mice with Impaired Acquisition of Gait Function Caused by Neonatal Brain Injury

Figure 1A shows the experimental schedule of brain injury^17,18) and habilitation. The habilitation applied is termed air-walk habilitation and consisted of a daily 20-min session in which mice were placed in a rubber band and suspended without floor grounding to promote active limb movements that mimic gait behaviors (Fig. 1B).

First, to examine the effects of injury and habilitation on mouse growth, we measured body weight at 1–4 weeks post-injury (wpi) on days P9, 16, 23, and 30. There were no significant differences of body weight at 1–4 wpi among the control, injury, and habilitation groups, suggesting that injury and habilitation did not affect overall body growth (Fig. 1C). Next, the brain injury volume was measured at 0.5 and 4 wpi to examine the effect of habilitation on the recovery process of injured brain tissue. Injury volume was defined as the area inside the region with GFAP-positive signals, which indicate the glial scar area. Injury volume at 0.5 and 4 wpi was not affected by habilitation intervention (Fig. 1D, E), suggesting that habilitation does not affect the brain injury volume.

Mice begin quadrupedal locomotion at 2 weeks old and acquire a gait pattern similar to that of adults at 4 weeks old, after weaning.²⁰⁾ To investigate the effects of brain injury and habilitation on this gait development process, the Foot Fault Test,²¹⁾ in which missteps were recorded if the mouse slipped or fell with one of its limbs dropping into an opening in the grid, was conducted at 2 and 4 wpi (Fig. 2A). While the left–right ratio of foot faults is 50:50 in normal mice, the left foot-fault ratio increases if the right hemisphere of the brain is injured. Consistent with a previous report,¹⁸⁾ the left foot-fault ratio increased in cryogenically injured mice at both 2 and 4 wpi, indicating that gait function was impaired in neonatal brain-injured mice (Fig. 2B). In the habilitation group, the increase in the left foot-fault ratio as a result of injury was significantly reduced at 2 and 4 wpi (Fig. 2B). Together, these results suggest that habilitation promotes motor learning of gait function in mice with impaired acquisition of gait function caused by neonatal brain injury.

Fig. 2.

Habilitation overcomes impaired gait function following neonatal brain injury. (A) Experimental scheme. Foot fault tests were performed at 2 and 4 wpi (yellow arrowheads) in the control, injury, and habilitation groups. (B) Percentage of left limb faults to total limb faults at 2 and 4 wpi in control, injury, and habilitation groups. *P<0.05. Data shown are mean ± SEM.

Developmental Process of Normal Gait Function in Mice

During the process of human gait development, the swing function of the leg at the hip joint develops first, followed by maturation of the stance function, e.g., knee and ankle flexion and plantar function in the stance phase, resulting in acquisition of mature gait function in both the swing and stance phases.²²⁾ In mice with mature gait function, gait behaviors are also divided into the stance and swing phases; however, the detailed developmental processes involved are unknown. To investigate gait development in mice, we used the Catwalk XT system. This system enables objective and quantitative analysis of gait elements and has been used in previous studies to detect gait dysfunction in stroke and spinal cord injury in adult mice.^13,14) We identified five parameters (body speed, swing speed, max contact max intensity, max contact mean intensity, and print area) useful for quantitatively assessing the process of normal gait acquisition in developing mice. These parameters could be evaluated by adjusting the camera sensitivity and illumination brightness of the Catwalk XT system.

First, to evaluate the overall development of gait, we analyzed body speed (Fig. 3A, B), a parameter indicating limb speed during the swing and stance phases. We found that the body speed continuously increased from 2 to 4 wpi (Fig. 3C-F, blue) in uninjured control mice, suggesting that the gait function develops continuously.

Fig. 3.

Changes in body speed during postnatal gait development in the control, injury, and habilitation groups. (A, B) Schematic of body speed analysis used in this study. (C-F) Body speed of the right forelimb (C), left forelimb (D), right hindlimb (E), and left hindlimb (F) at 2, 3, and 4 wpi in control, injury, and habilitation groups. *P<0.05. Data shown are mean ± SEM.

During human gait development, the swing speed of the legs during the swing phase increases with development.²³⁾ We analyzed the swing speed (Fig. 4A, B) in mice to see whether similar changes occur during gait development. In control mice, we found that the swing speed significantly increased from 2 to 3 wpi and then remained unchanged at 4 wpi (Fig. 4C-F, blue), suggesting that the swing phase gait function matures early in the gait development process.

Fig. 4.

Changes in swing speed during postnatal gait development in the control, injury, and habilitation groups. (A, B) Schematic of swing speed analysis used in study. (C-F) Swing speed of the right forelimb (C), left forelimb (D), right hindlimb (E), and left hindlimb (F) at 2, 3, and 4 wpi in control, injury, and habilitation groups. *P<0.05. Data shown are mean ± SEM.

During human gait development, the pressure and print area at the maximum ground contact during the stance phase decreases because of the formation of the plantar foot.²²⁾ To investigate whether ground contact pressure also decreases during gait development in mice, we evaluated the plantar ground pressure at 2, 3, and 4 wpi (Fig. 5A). The mean (max contact mean intensity) and maximum (max contact max intensity) pressures at maximum ground contact were used as parameters reflecting plantar ground pressure when full body weight was applied to the ankle joint. We found that both max contact mean intensity and max contact max intensity decreased between 3 and 4 wpi in control mice (Fig. 5B-I, blue). Furthermore, a decrease in print area at maximum ground contact was also observed during this period (Fig. S1). Together, these results suggest that maturation of the pressure and area at maximum ground contact in the stance phase, which occurs in humans, also occurs in mice.

Fig. 5.

Changes in maximum and mean contact intensities during postnatal gait development in the control, injury, and habilitation groups. (A) Schematic of maximum ground contact pressure and mean maximum ground contact pressure used in this analysis. (B-I) Max contact mean intensity (B-E) and max contact max intensity (F-I) of the right forelimb (B, F), left forelimb (C, G), right hindlimb (D, H) and left hindlimb (E, I) at 2, 3, and 4 wpi in control, injury, and habilitation groups. ODU, optical density unit. *P<0.05, **P<0.01. Data shown are mean ± SEM.

Figure S1.

Changes in print area during normal gait development in mice. (A-D) Changes in total print area of the right forelimb (A), left forelimb (B), right hindlimb (C) and left hindlimb (D) at 2, 3, and 4 wpi in control (no injury) mice. (E-H) Changes in finger and palm print area of the right forelimb (E), left forelimb (F), right hindlimb (G) and left hindlimb (H) at 3 and 4 wpi in control mice. *P<0.05; **P<0.01. Data shown are mean ± SEM.

Our results suggest that the Catwalk XT system can be used to quantitatively evaluate the process of mouse gait development. Furthermore, these results suggest that, as in humans, the stance phase function matures after the swing phase function during gait development in mice.

Neonatal Brain Injury Impairs Normal Gait Development in Mice

To investigate how neonatal brain injury impairs gait function (Fig. 2), we quantitatively examined gait development in brain-injured mice at 2–4 wpi using Catwalk XT. We found that the continuous increase in body speed observed in normal development is impaired by the injury (Fig. 3C-F, red). Next, gait development in the swing and stance phases was compared between normal and injured mice. In the swing phase, an increase in swing speed was observed at 2–3 wpi in the control group, whereas such an increase was observed at 3–4 wpi in the injured group (Fig. 4C-F, red), suggesting that the functional maturation in the swing phase was delayed. In the stance phase, the decrease in max contact mean intensity and max contact max intensity observed at 3–4 wpi in the control group was not observed in the injured group (Fig. 5B-I, red), suggesting that functional maturation in the stance phase was impaired. These results suggest that neonatal brain injury impairs and delays the function of the stance and swing phases, respectively, thereby impairing the acquisition of gait function.

Effects of Habilitation on Recovery of Gait Developmental Deficits in Neonatal Brain-injured Mice

The air-walk habilitation performed in this study (Fig. 1B) may stimulate swing-phase function because it promotes leg swing. However, it is unclear whether such habilitation affects function in the stance phase because the mice are suspended without any loading. To investigate the mechanism by which habilitation restores injury-induced gait deficits (Fig. 2), we analyzed gait development in mice in the habilitation group at 2–4 wpi using Catwalk XT. We found that body speed increased from 2 to 3 wpi and remained unchanged at 4 wpi (Fig. 3C-F, green). In the swing phase, an increase in swing speed was observed in the habilitation group from 2 to 3 wpi, just as it is in the normal group (Fig. 4C-F, green). However, in the stance phase, in the habilitation group, as in the injury group, neither the max contact mean intensity nor max contact max intensity decreased between 3 and 4 wpi (Fig. 5B-I, green). Taken together, these results suggest that the habilitation provided in this study promoted motor learning of gait function in the swing phase.

DISCUSSION

Previous reports have analyzed gait deficits in stroke and spinal cord injury models in adult mice.^24,25) Gait deficits in white matter injury and spinocerebellar degeneration models using neonatal mice have also been reported.^26,27) However, the details of the gait acquisition process in mice and injury-induced gait abnormalities during the postnatal period are unknown. In the present study, by modifying parameters of the Catwalk XT system, which is usually used for gait analysis in adult mice, we clarified the normal gait development process in mice and elucidated the effects of brain injury and habilitation on the gait development process. Our results serve as a basis for the analysis of gait function in mice under physiological and pathological conditions.

In the present analysis, we found an increase in swing speed in 2- to 3-week-old mice and a decrease in plantar ground pressure in 3- to 4-week-old mice under physiological conditions (Fig. 4, 5). In human gait development, it is known that the speed of leg swing at the hip joint develops first, followed by a decrease in plantar ground pressure, which is known as the acquisition of a rocker function.²²⁾ Consequently, our results suggest the existence of a maturation mechanism of gait function in mice similar to the well-known acquisition of the rocker function in humans.

During the maturation of gait function in mice, the print area at the point of maximum ground contact is expected to increase with limb growth. However, we found that the area showed a significant decrease in 4-week-old mice compared with 3-week-old mice (Fig. S1). In humans, tarsal bones are lifted by the development of the lower leg muscles, resulting in a decrease in the foot area and the acquisition of the rocker function.²²⁾ Therefore, it is possible that in mice, as in humans, the reduction in finger and palm area at maximum ground contact contributes to the maturation of rocker-like gait function.

Neonatal cryogenic injury in the cerebral cortex impaired and delayed the development of functions in the stance and swing phases, respectively, thereby resulting in impaired development of gait speed (Fig. 4, 5). However, the brain injury model used in this study did not affect the brainstem or spinal cord. Therefore, the pattern generator was not likely to have been affected, and no impairment of gait pattern or rhythm was observed.¹⁸⁾ Consequently, the gait dysfunction observed in this injury model is likely the result of delayed maturation of the rocker function.

By continuously promoting active locomotion in the swing phase from the early post-injury period, the unloaded habilitation performed in this study improved injury-induced gait dysfunction even under loaded conditions (Fig. 2). These results suggest that habilitation to promote active locomotion may improve gait function in human neonates following brain injury. CatWalk analysis showed that habilitation reduced the injury-induced delay in maturation of the swing function (Fig. 4). Because sensory input may beneficially affect motor learning,²⁸⁾ further restoration of the impaired rocker function in combination with habilitation to support the development of ground contact pressure may be necessary for more effective improvement of gait function in the future.

The detailed mechanism of recovery of gait function by the habilitation performed in the current study remains unclear. Cortical lesions, mainly in the primary motor cortex and somatosensory cortex, result in impairment of motor function governed by the corticospinal tracts from these areas. The fact that the habilitation in this study was an active movement that mimicked the movement during the swing phase suggests that it is possible that promotion of the development of proximal limb-muscle function contributed to the increase in swing speed. Furthermore, dendritic and synaptic reorganization in the neural circuitry surrounding the lesion²⁹⁾ and/or regeneration of neurons produced in the ventricular-subventricular zone^18,30) may also have contributed to the gait improvement. A recent study reported that resting-state functional magnetic resonance imaging (rs-fMRI) can be used to assess cortical plasticity after spinal cord injury in adult mice.³¹⁾ Therefore, use of rs-fMRI to examine the effects of neonatal brain injury and habilitation may reveal the detailed mechanisms of functional gait improvement. Because brain plasticity^6,7) and neurogenic capacity^8,9) are higher in the neonatal period than in the adult, it is important to analyze the mechanisms of habilitation by active movements at the cellular and molecular levels in mice and in humans.

This study provides a basis for gait development research using mice. Moreover, our findings of the improvement of injury-induced impaired gait function by habilitation initiated during the neonatal period suggest possible new treatments and habilitation interventions for poor gait development caused by neonatal brain injury. Furthermore, this study may serve as a basis for future research on the treatment of gait impairment after hypoxic–ischemic encephalopathy and periventricular leukomalacia in human neonates.

ACKNOWLEDGMENTS

We thank Ikuo Wada for critical reading of the manuscript, Hideo Jinnou and the Center for Experimental Animal Science in Nagoya City University for technical support, and Sawamoto laboratory members for discussions. This work was supported by research grants from the Japan Agency for Medical Research and Development [22gm1210007 (to K.S. )], the Japan Society for the Promotion of Science KAKENHI [20H05700 (to K.S.) and 21K06395 (to M.S.)], a Grant-in-Aid for Research at Nagoya City University (to K.S. and M.S.), the Canon Foundation (to K.S.), and the Takeda Science Foundation (to K.S.).

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no conflicts of interest.

REFERENCES

• 1.  Oskoui M, Coutinho F, Dykeman J, Jetté N, Pringsheim T: An update on the prevalence of cerebral palsy: a systematic review and meta-analysis. Dev Med Child Neurol 2013;55:509–519. PMID:23346889, DOI:10.1111/dmcn.12080
• 2.  Kawamoto H, Kamibayashi K, Nakata Y, Yamawaki K, Ariyasu R, Sankai Y, Sakane M, Eguchi K, Ochiai N: Pilot study of locomotion improvement using hybrid assistive limb in chronic stroke patients. BMC Neurol 2013;13:141. PMID:24099524, DOI:10.1186/1471-2377-13-141
• 3.  Dodd KJ, Foley S: Partial body-weight-supported treadmill training can improve walking in children with cerebral palsy: a clinical controlled trial. Dev Med Child Neurol 2007;49:101–105. PMID:17253995, DOI:10.1111/j.1469-8749.2007.00101.x
• 4.  Kaneko F, Yasojima T, Kizuka T: Kinesthetic illusory feeling induced by a finger movement movie effects on corticomotor excitability. Neuroscience 2007;149:976–984. PMID:17935897, DOI:10.1016/j.neuroscience.2007.07.028
• 5.  Campbell SK, Cole W, Boynewicz K, Zawacki LA, Clark A, Gaebler-Spira D, deRegnier RA, Kuroda MM, Kale D, Bulanda M, Madhavan S: Behavior during tethered kicking in infants with periventricular brain injury. Pediatr Phys Ther 2015;27:403–412. PMID:26397087, DOI:10.1097/PEP.0000000000000173
• 6.  Wolpaw JR: Harnessing neuroplasticity for clinical applications. Brain 2012;135:e215, author reply e216. PMID:22374936, DOI:10.1093/brain/aws017
• 7.  Kolb B, Harker A, Gibb R: Principles of plasticity in the developing brain. Dev Med Child Neurol 2017;59:1218–1223. PMID:28901550, DOI:10.1111/dmcn.13546
• 8.  Sanai N, Nguyen T, Ihrie RA, Mirzadeh Z, Tsai HH, Wong M, Gupta N, Berger MS, Huang E, Garcia-Verdugo JM, Rowitch DH, Alvarez-Buylla A: Corridors of migrating neurons in the human brain and their decline during infancy. Nature 2011;478:382–386. PMID:21964341, DOI:10.1038/nature10487
• 9.Paredes MF, James D, Gil-Perotin S, Kim H, Cotter JA, Ng C, Sandoval K, Rowitch DH, Xu D, McQuillen PS, Garcia-Verdugo JM, Huang EJ, Alvarez-Buylla A. Extensive migration of young neurons into the infant human frontal lobe. Science 2016;354:aaf7073. PMID:27846470, DOI:10.1126/science.aaf7073
• 10.  Vandeputte C, Taymans JM, Casteels C, Coun F, Ni Y, Van Laere K, Baekelandt V: Automated quantitative gait analysis in animal models of movement disorders. BMC Neurosci 2010;11:92. PMID:20691122, DOI:10.1186/1471-2202-11-92
• 11.  Deacon RM: Measuring motor coordination in mice. J Vis Exp 2013; e2609. PMID:23748408, DOI:10.3791/2609
• 12.  Ådén U, Dahlberg V, Fredholm BB, Lai LJ, Chen Z, Bjelke B: MRI evaluation and functional assessment of brain injury after hypoxic ischemia in neonatal mice. Stroke 2002;33:1405–1410. PMID:11988622, DOI:10.1161/01.STR.0000014608.78503.DB
• 13.  Neumann M, Wang Y, Kim S, Hong SM, Jeng L, Bilgen M, Liu J: Assessing gait impairment following experimental traumatic brain injury in mice. J Neurosci Methods 2009;176:34–44. PMID:18805438, DOI:10.1016/j.jneumeth.2008.08.026
• 14.  Gensel JC, Tovar CA, Hamers FP, Deibert RJ, Beattie MS, Bresnahan JC: Behavioral and histological characterization of unilateral cervical spinal cord contusion injury in rats. J Neurotrauma 2006;23:36–54. PMID:16430371, DOI:10.1089/neu.2006.23.36
• 15.  Koopmans GC, Brans M, Gómez-Pinilla F, Duis S, Gispen WH, Torres-Aleman I, Joosten EA, Hamers FP: Circulating insulin-like growth factor I and functional recovery from spinal cord injury under enriched housing conditions. Eur J Neurosci 2006;23:1035–1046. PMID:16519668, DOI:10.1111/j.1460-9568.2006.04627.x
• 16.  Krug H, Dorman C, Frizelle S, Mahowald M, Funkenbusch S: A comparison of DigiGait™ and TreadScan™ imaging systems: assessment of pain using gait analysis in murine monoarthritis. J Pain Res 2013;7:25–35. PMID:24516338, DOI:10.2147/JPR.S52195
• 17.  Ajioka I, Jinnou H, Okada K, Sawada M, Saitoh S, Sawamoto K: Enhancement of neuroblast migration into the injured cerebral cortex using laminin-containing porous sponge. Tissue Eng Part A 2015;21:193–201. PMID:25010638, DOI:10.1089/ten.tea.2014.0080
• 18.  Jinnou H, Sawada M, Kawase K, Kaneko N, Herranz-Pérez V, Miyamoto T, Kawaue T, Miyata T, Tabata Y, Akaike T, García-Verdugo JM, Ajioka I, Saitoh S, Sawamoto K: Radial glial fibers promote neuronal migration and functional recovery after neonatal brain injury. Cell Stem Cell 2018;22:128–137.e9. PMID:29276142, DOI:10.1016/j.stem.2017.11.005
• 19.  Sawada M, Ohno N, Kawaguchi M, Huang S, Hikita T, Sakurai Y, Bang Nguyen H, Quynh Thai T, Ishido Y, Yoshida Y, Nakagawa H, Uemura A, Sawamoto K: PlexinD1 signaling controls morphological changes and migration termination in newborn neurons. EMBO J 2018;37:e97404. PMID:29348324, DOI:10.15252/embj.201797404
• 20.  Akula SK, McCullough KB, Weichselbaum C, Dougherty JD, Maloney SE: The trajectory of gait development in mice. Brain Behav 2020;10:e01636. PMID:32333523, DOI:10.1002/brb3.1636
• 21.  Barth TM, Jones TA, Schallert T: Functional subdivisions of the rat somatic sensorimotor cortex. Behav Brain Res 1990;39:73–95. PMID:2390194, DOI:10.1016/0166-4328(90)90122-U
• 22.  Sutherland DH, Olshen R, Cooper L, Woo SL: The development of mature gait. J Bone Joint Surg Am 1980;62:336–353. PMID:7364807, DOI:10.2106/00004623-198062030-00004
• 23.  Preis S, Klemms A, Müller K: Gait analysis by measuring ground reaction forces in children: changes to an adaptive gait pattern between the ages of one and five years. Dev Med Child Neurol 1997;39:228–233. PMID:9183260, DOI:10.1111/j.1469-8749.1997.tb07416.x
• 24.  Pollak J, Doyle KP, Mamer L, Shamloo M, Buckwalter MS: Stratification substantially reduces behavioral variability in the hypoxic–ischemic stroke model. Brain Behav 2012;2:698–706. PMID:23139913, DOI:10.1002/brb3.77
• 25.  Apostolova I, Irintchev A, Schachner M: Tenascin-R restricts posttraumatic remodeling of motoneuron innervation and functional recovery after spinal cord injury in adult mice. J Neurosci 2006;26:7849–7859. PMID:16870730, DOI:10.1523/JNEUROSCI.1526-06.2006
• 26.  Zaghloul N, Kurepa D, Bader MY, Nagy N, Ahmed MN: Prophylactic inhibition of NF-κB expression in microglia leads to attenuation of hypoxic ischemic injury of the immature brain. J Neuroinflammation 2020;17:365. PMID:33261624, DOI:10.1186/s12974-020-02031-9
• 27.  Cantoria MJ, See PA, Singh H, de Leon RD: Adaptations in glutamate and glycine content within the lumbar spinal cord are associated with the generation of novel gait patterns in rats following neonatal spinal cord transection. J Neurosci 2011;31:18598–18605. PMID:22171058, DOI:10.1523/JNEUROSCI.3499-11.2011
• 28.  Davies PL, Chang WP, Gavin WJ: Maturation of sensory gating performance in children with and without sensory processing disorders. Int J Psychophysiol 2009;72:187–197. PMID:19146890, DOI:10.1016/j.ijpsycho.2008.12.007
• 29.  Nudo RJ, Wise BM, SiFuentes F, Milliken GW: Neural substrates for the effects of rehabilitative training on motor recovery after ischemic infarct. Science 1996;272:1791–1794. PMID:8650578, DOI:10.1126/science.272.5269.1791
• 30.  Liang H, Zhao H, Gleichman A, Machnicki M, Telang S, Tang S, Rshtouni M, Ruddell J, Carmichael ST: Region-specific and activity-dependent regulation of SVZ neurogenesis and recovery after stroke. Proc Natl Acad Sci USA 2019;116:13621–13630. PMID:31196958, DOI:10.1073/pnas.1811825116
• 31.  Matsubayashi K, Nagoshi N, Komaki Y, Kojima K, Shinozaki M, Tsuji O, Iwanami A, Ishihara R, Takata N, Matsumoto M, Mimura M, Okano H, Nakamura M: Assessing cortical plasticity after spinal cord injury by using resting-state functional magnetic resonance imaging in awake adult mice. Sci Rep 2018;8:14406. PMID:30258091, DOI:10.1038/s41598-018-32766-8