Citation: Battistini, J.I.; Mastrorilli, V.;
Nicolis di Robilant, V.; Saraulli, D.;
Marinelli, S.; Farioli Vecchioli, S. Role
of Running-Activated Neural Stem
Cells in the Anatomical and
Functional Recovery after Traumatic
Brain Injury in p21 Knock-Out Mice.
Int. J. Mol. Sci. 2023, 24, 2911.
https://doi.org/10.3390/ijms24032911
Academic Editors: Daniele Bottai
and Ola Hermanson
Received: 8 December 2022
Revised: 28 January 2023
Accepted: 30 January 2023
Published: 2 February 2023
Copyright:
© 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
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Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
 
International Journal of 
Molecular Sciences
Article
Role of Running-Activated Neural Stem Cells in the
Anatomical and Functional Recovery after Traumatic Brain
Injury in p21 Knock-Out Mice
Jonathan Isacco Battistini 1
, Valentina Mastrorilli 2, Vittoria Nicolis di Robilant 3, Daniele Saraulli 4,
Sara Marinelli 1
and Stefano Farioli Vecchioli 1,*
1
Institute of Biochemistry and Cell Biology, Institute of Biochemistry and Cell Biology,
National Research Council (IBBC/CNR), Monterotondo, 00015 Rome, Italy
2
Plaisant S.R.L., 00128 Rome, Italy
3
Experimental Translational Oncology Department at Menarini Ricerche, Pomezia, 00071 Rome, Italy
4
Department of Law, Economics, Politics and Modern Languages, LUMSA University, 00193 Rome, Italy
*
Correspondence: stefano.fariolivecchioli@cnr.it
Abstract: Traumatic brain injury (TBI) represents one of the most common worldwide causes of death
and disability. Clinical and animal model studies have evidenced that TBI is characterized by the loss
of both gray and white matter, resulting in brain atrophy and in a decrease in neurological function.
Nowadays, no effective treatments to counteract TBI-induced neurological damage are available. Due
to its complex and multifactorial pathophysiology (neuro-inﬂammation, cytotoxicity and astroglial
scar formation), cell regeneration and survival in injured brain areas are strongly hampered. Recently,
it has been proposed that adult neurogenesis may represent a new approach to counteract the post-
traumatic neurodegeneration. In our laboratory, we have recently shown that physical exercise
induces the long-lasting enhancement of subventricular (SVZ) adult neurogenesis in a p21 (negative
regulator of neural progenitor proliferation)-null mice model, with a concomitant improvement of
olfactory behavioral paradigms that are strictly dependent on SVZ neurogenesis. On the basis of this
evidence, we have investigated the effect of running on SVZ neurogenesis and neurorepair processes
in p21 knock-out mice that were subject to TBI at the end of a 12-day session of running. Our data
indicate that runner p21 ko mice show an improvement in numerous post-trauma neuro-regenerative
processes, including the following: (i) an increase in neuroblasts in the SVZ; (ii) an increase in the
migration stream of new neurons from the SVZ to the damaged cortical region; (iii) an enhancement
of new differentiating neurons in the peri-lesioned area; (iv) an improvement in functional recovery
at various times following TBI. All together, these results suggest that a running-dependent increase
in subventricular neural stem cells could represent a promising tool to improve the endogenous
neuro-regenerative responses following brain trauma.
Keywords: subventricular zone; adult neurogenesis; neural stem cells; traumatic brain injury; p21
1. Introduction
Traumatic brain injury (TBI) is one of the most common causes of death and disability
in young people [1]. TBI severity is assessed on the basis of the Glasgow Coma Scale (GCS)
score [2], with further modiﬁcations and can be graded as mild, moderate or severe [3,4].
The symptoms of mild patients are short-term memory and concentration difﬁculties and
they usually show complete neurological recovery [5]. Moderate patients are lethargic
and stuporous, whilst severe subjects are comatose, unable to open their eyes or follow
commands [6]. Severe patients also have a higher risk of hypotension, hypoxemia and brain
swelling, all effects that, if not prevented, can cause complications and lead to death [7,8].
Moreover, TBI strongly increases a patient’s susceptibility to neurodegenerative diseases
such as Alzheimer’s and Parkinson’s disease [9,10].
Int. J. Mol. Sci. 2023, 24, 2911. https://doi.org/10.3390/ijms24032911
https://www.mdpi.com/journal/ijms
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The adult mammalian brain is able to respond to damage with structural and func-
tional modiﬁcations and regeneration mechanisms. In this context, the role of the endoge-
nous neurogenic post-traumatic response is the subject of a wide range of studies [11,12].
Under physiological conditions, the neural stem cells (NSCs, named type B cells) that reside
in the SVZ represent a population of relatively quiescent cells [13,14], which give rise, in
the course of neurogenic differentiation, to the following two classes of cells: type C cells
that have a high proliferative rate [15], which in turn give rise to neuroblasts called type A
cells [16]. These cells exit the cell cycle and migrate along the rostral migratory stream to
reach the olfactory bulb, where they mature into inhibitory GABAergic neurons [17,18]. Af-
ter brain injury, endogenous neural stem cells can be activated to modulate the timing and
rate of proliferation/differentiation, with the aim of facilitating brain repair [19,20]. This
process is also ﬁnely regulated by a series of changes in the environment of the neurogenic
niche, such as an increase in vasculature permeability that favors the migration of NSCs
and neuroblasts to the injured cortex [21]. Furthermore, transcriptomic studies based on
single-cell RNA sequencing (RNA-seq) have shown a strong increase in the post-injured
SVZ of the proportion of “primed” quiescent and active NSCs, which highly express genes
related to protein synthesis and cell cycle regulation [22]. These ﬁndings highlighted the
existence of reactive SVZ NSCs capable of conferring protection following TBI, suggesting
an important role of the activation of SVZ NSCs in providing beneﬁcial outcomes for
post-TBI brain repair.
The p21Waf1/Cip1 gene represents one of the main regulators of the cell cycle and plays
a primary role in modulating the transition between quiescence and activation of NSCs in
adult neurogenic niches [23]. This gene is part of the Cip/Kip family of cyclin-dependent
kinase inhibitors (CKIs), which also include the p27 and p57 genes with the function of neg-
atively regulating cell cycle progression [24,25]. Within neurogenic niches, p21 expression
correlates with the maintenance of NSCs in a quiescent state and with the restraining of
progenitor proliferation [26–28]. Constitutive deletion of the p21 gene in mouse models
has led to the rapid and powerful activation in the cell cycle of quiescent NSCs at the
post-natal stage, with a consequent reduction in the self-renewal capacity of NSCs, the
onset of replicative stress in the hyper-proliferating NSCs and progenitors and a reduction
in neurogenesis in adult mice [28–31]. Previous work by our group demonstrated that in
the absence of the p21 gene, SVZ neurogenesis and olfactory behavior are signiﬁcantly
enhanced by 12 days of voluntary running. These results strongly indicate that p21-null
NSCs retain their high neurogenic potential, which is speciﬁcally triggered by physical
activity [28]. Based on these ﬁndings, in this project, we investigated the possibility that the
enhancement of SVZ neurogenesis that occurs in a p21-null mouse model that undergoes a
12 day-session of voluntary running could be an effective mechanism that contributes to the
neuroanatomical and functional recovery processes following pathological conditions such
as TBI. Our results demonstrate that at different time-points following TBI, the combination
of running and of p21 knockdown induces an increase in the migration of SVZ NSCs and
progenitors toward the cortical lesion, an enhancement of new differentiating neurons
in the peri-lesioned area and partial functional recovery in the injured mice. These data
suggest a potential model for the strengthening of post-traumatic endogenous neurogenesis
that is capable of effectively counteracting the anatomical and functional dysfunctions
induced by cortical damage and accelerating neurorepair processes.
2. Results
The aim of this work is to analyze the post-traumatic neurogenic effects of a running
session of 12 days in a mouse model with the deletion of p21. The 12-day running paradigm
was chosen in accordance with previous data that demonstrate that this running protocol
provokes the peak of running-dependent increments of neurogenesis in the SVZ of p21 ko
mice [28]. The experimental procedure is detailed in the Material and Method section and
is shown in Figure 1.
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paradigm was chosen in accordance with previous data that demonstrate that this 
running protocol provokes the peak of running-dependent increments of neurogenesis in 
the SVZ of p21 ko mice [28]. The experimental procedure is detailed in the Material and 
Method section and is shown in Figure 1. 
 
Figure 1. Graphical representation of experimental procedures. (A) Experimental timeline. The 
animals have been subjected to a running session for 12 days. From day 7 of the running session 
until the day of the lesion, BrdU is administered in the drinking water of the animals. On the 12th 
day, the mice undergo the CCI surgical procedure. The functional outcome has been evaluated by 
the Ladder Rung Walking Task (LWT) 24 h before CCI (pre-TBI) to determine the baseline number 
of errors, and two, seven, fourteen and thirty-three days after the surgery (P2, P7, P14 and P30). At 
P7, P14 and P30, the animals have been sacrificed to perform immunofluorescence assays (IF). (B) 
Figure 1. Graphical representation of experimental procedures. (A) Experimental timeline. The
animals have been subjected to a running session for 12 days. From day 7 of the running session until
the day of the lesion, BrdU is administered in the drinking water of the animals. On the 12th day, the
mice undergo the CCI surgical procedure. The functional outcome has been evaluated by the Ladder
Rung Walking Task (LWT) 24 h before CCI (pre-TBI) to determine the baseline number of errors, and
two, seven, fourteen and thirty-three days after the surgery (P2, P7, P14 and P30). At P7, P14 and
P30, the animals have been sacriﬁced to perform immunoﬂuorescence assays (IF). (B) Coronal section
of a lesioned brain. To evaluate the cellular processes that occur in the brain after the trauma, we
considered the following: the ipsi- and contralateral SVZ, the migratory route and the low, medial
and lateral sides of the lesioned cortex. Scale bar = 400 µm.
Int. J. Mol. Sci. 2023, 24, 2911
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2.1. TBI Induces the Signiﬁcant Activation of Type B NSCs in the SVZ of Injured Mice
To understand the impact of TBI on type B Glia-like NSC recruitment and proliferation,
we analyze, at different time points from the trauma (7, 14 and 30 days), the ipsi- and
contralateral SVZ of mice subjected to brain injury or in the SHAM condition. The NSC
sub-population was identiﬁed through the co-localization of the GFAP marker and the
Nestin GFP transgene.
The data show a strong increase in the SVZ of both hemispheres of type B cell recruit-
ment from quiescence in the TBI groups, in comparison with their SHAM counterpart at
7 days post TBI (recruitment: ratio of Ki67+NestinGFP+GFAP+ cells/NestinGFP+GFAP+
total cells, ipsilateral: p < 0.001, Figure 2A–C, Supplementary Figure S1A–H; contralateral:
p < 0.001, Supplementary Figure S2A) and their proliferation (Ki67+NestinGFP+GFAP+
cells, ipsilateral: p < 0.001, Figure 2A,B,D; contralateral: p < 0.001, Supplementary Figure S2B),
as well as 14 days after injury (recruitment: ipsilateral, p < 0.001, Figure 2E, Supplementary
Figure S3A–H; contralateral: p < 0.001, Supplementary Figure S2C; proliferation: ipsilateral:
p < 0.001, Figure 2F, Supplementary Figure S3A–H; contralateral: p < 0.001, Supplementary
Figure S2D).
After 30 days from TBI, we observe in the SVZ of both hemispheres a main lesion
effect of the type B cells’ pool of TBI groups compared to the SHAM animals, due to the
strong increase observed in the KO TBI and KO RUN TBI mice, compared to the respective
SHAM mice (NestinGFP+GFAP+ cells, ipsilateral: p < 0.001, Figure 2G, Supplementary
Figure S4A–H; contra-lateral: p = 0.002, Supplementary Figure S2E).
These data let us hypothesize that TBI might induce the powerful activation of type B
cells, in terms of recruitment from quiescence and proliferation, leading in the long term to
an expansion of the pool compared to the SHAM groups.
2.2. Inﬂuence of p21 Deletion and Running Session on TBI-Induced NSC Activation
Moreover, we evaluated the different responses to trauma within the injured groups,
with the aim of assessing whether the deletion of p21 and/or the physical activity before
the TBI might promote the neurogenic response. The comparative analysis within the
four TBI groups (WT TBI, WT RUN TBI, KO TBI and KO RUN TBI) indicates that at
7 days post trauma, no changes were detectable in the recruitment or expansion of type
B cells. Instead, after 14 days, we observe, in the contralateral SVZ, a TBI-dependent
expansion of type B cells in the KO RUN TBI, with respect to the other groups in terms of
the enhancement of type B proliferation (KO RUN TBI vs. WT TBI and KO TBI, p < 0.001,
Supplementary Figure S2D). In addition, 30 days after the TBI, in the ipsilateral SVZ, no
signiﬁcant variations were detected, while in the contralateral SVZ, a strong expansion of
type B pool size in the KO RUN TBI mice was detected (NestinGFP+GFAP+ cells: KO RUN
TBI vs. WT TBI, WT RUN TBI and KO TBI, p < 0.001, Supplementary Figure S2E).
From this ﬁrst analysis, it emerges that the deletion of p21 and/or physical activity
is ineffective in triggering the post-traumatic NSC response in the ipsilateral hemisphere,
while a strong effect of running and p21 deletion on NSC activation is observed in the
contralateral hemisphere.
2.3. Time-Course of Neural Stem Progenitor Cell (NSPC) Modulation after TBI
The count of NestinGFP+ cells and their proliferating fraction (Ki67+ NestinGFP+
cells) allowed us to evaluate the variations in the stem cells and the transit amplifying
progenitors (small fraction of type B and C cells), hereinafter collectively referred to as
neural stem/progenitor cells (NSPCs).
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Figure 2. Modulation of type B cells after TBI. (A,B) Representative images in coronal sections of the 
neurogenic processes that occur in the ipsilateral SVZ of p21 ko mice 7 days after TBI (KO TBI, (A)) 
and after SHAM (KO SHAM, (B)). The images show an increment in the pool of proliferating type 
B cells (Ki67+/GFAP+/NestinGFP+ cells) in the KO TBI animals, with respect to the KO SHAM mice 
at 7 days after the surgery (N = 5 mice/group). (C) Graph shows the increase in type B recruitment 
at 7 days post TBI in the ipsilateral SVZ of the mice subjected to injury in comparison to their SHAM 
groups (ratio of Ki67+ NestinGFP+ GFAP+ cells/NestinGFP+ GFAP+ total cells, ipsilateral: lesion effect: 
F(1,60) = 91.45, p < 0.001). (D) Graph shows the enhancement of type B proliferation in the ipsi-lateral 
SVZ of TBI groups (Ki67+ NestinGFP+ GFAP+ cells, ipsilateral: lesion effect: F(1,60) = 45.8 p < 0.001). (E) 
Histograms illustrate the increase in type B recruitment in the ipsilateral SVZ in mice after 14 days 
from TBI (lesion effect: F(1,56) = 188, p < 0.001, $). (F) Graphs indicate an increase in type B proliferation 
in the ipsi-lateral SVZ of mice subjected to TBI 14 days after the trauma (lesion effect: F(1,56) = 85.9, p 
< 0.001). (G) After 30 days from TBI, we observed a significant increase in the type B cell population 
in the TBI mice with respect to their SHAM groups (NestinGFP+ GFAP+ cells, ipsilateral: lesion effect: 
F(1,44) = 21.76, p < 0.001, $). Statistical significance of main lesion effect between SHAM and TBI 
Figure 2. Modulation of type B cells after TBI. (A,B) Representative images in coronal sections of the
neurogenic processes that occur in the ipsilateral SVZ of p21 ko mice 7 days after TBI (KO TBI, (A))
and after SHAM (KO SHAM, (B)). The images show an increment in the pool of proliferating type B
cells (Ki67+/GFAP+/NestinGFP+ cells) in the KO TBI animals, with respect to the KO SHAM mice at
7 days after the surgery (N = 5 mice/group). (C) Graph shows the increase in type B recruitment at
7 days post TBI in the ipsilateral SVZ of the mice subjected to injury in comparison to their SHAM
groups (ratio of Ki67+ NestinGFP+ GFAP+ cells/NestinGFP+ GFAP+ total cells, ipsilateral: lesion
effect: F(1,60) = 91.45, p < 0.001). (D) Graph shows the enhancement of type B proliferation in the
ipsi-lateral SVZ of TBI groups (Ki67+ NestinGFP+ GFAP+ cells, ipsilateral: lesion effect: F(1,60) = 45.8
p < 0.001). (E) Histograms illustrate the increase in type B recruitment in the ipsilateral SVZ in mice
after 14 days from TBI (lesion effect: F(1,56) = 188, p < 0.001, $). (F) Graphs indicate an increase in type
B proliferation in the ipsi-lateral SVZ of mice subjected to TBI 14 days after the trauma (lesion effect:
F(1,56) = 85.9, p < 0.001). (G) After 30 days from TBI, we observed a signiﬁcant increase in the type B
Int. J. Mol. Sci. 2023, 24, 2911
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cell population in the TBI mice with respect to their SHAM groups (NestinGFP+ GFAP+ cells,
ipsilateral: lesion effect: F(1,44) = 21.76, p < 0.001, $). Statistical signiﬁcance of main lesion effect
between SHAM and TBI groups: $ p < 0.001. Multifactorial analysis with the following three
independent variables: genotype, treatment and running, followed by Fisher’s LSD post hoc tests.
Magniﬁcation = 20×. Scale bar = 100 µm. SVZ = subventricular zone. LV = lateral ventricle.
At 7 days post TBI, we observe in both ipsi- and contralateral regions a strong increase
in NSPC proliferation in the WT and KO groups subjected to TBI, with respect to their
SHAM groups (Ki67+NestinGFP+ cells, ipsilateral: WT TBI vs. WT SHAM and KO TBI vs.
KO SHAM p = 0.015, Figure 3A,C, Supplementary Figure S5A,B,E,F; contralateral: p = 0.013,
Supplementary Figure S6A), which induces a signiﬁcant increase in total proliferation
(Ki67+ cells, ipsilateral WT TBI vs. WT SHAM, p = 0.03, KO TBI vs. KO SHAM p < 0.001,
Figure 3A,D; contralateral: p = 0.0013, Supplementary Figure S6B). On the other hand,
in the KO RUN TBI group, we detect in the ipsi- and contralateral SVZ a net decrease,
compared to the KO RUN SHAM group, of proliferating NSPCs (Ki67+NestinGFP+ cells,
ipsilateral: KO RUN TBI vs. KO RUN SHAM, p = 0.009, Figure 3A,C, Supplementary
Figure S5G,H; contralateral: KO RUN TBI vs. KO RUN SHAM p < 0.001, Supplementary
Figure S6A) as well as of total proliferation (Ki67+ cells, ipsilateral: KO RUN TBI vs. KO
RUN SHAM, p < 0.001, Figure 3A,D, Supplementary Figure S5G,H; contralateral: KO RUN
TBI vs. KO RUN SHAM p < 0.001, Supplementary Figure S6B).
The analysis carried out 14 days post TBI demonstrates a signiﬁcant decrease in the
SVZ of both hemispheres of the NestinGFP+ cell pool in all the TBI groups compared
to their SHAM counterparts (ipsilateral: p < 0.001, Figure 3B,E; contralateral: p = 0.0011,
Supplementary Figure S6C). Moreover, we observe in the ipsilateral hemisphere of the
WT TBI and KO RUN TBI groups a sharp decrease in total proliferation, compared to the
respective WT SHAM and KO RUN SHAM groups (Ki67+ cells: WT TBI vs. WT SHAM,
p = 0.029; KO RUN TBI vs. KO RUN SHAM p < 0.001, Figure 3B,F), as well as in the number
of Ki67+ NestinGFP+ cells (WT TBI vs. WT SHAM, p = 0.06; KO RUN TBI vs. KO RUN
SHAM p = 0.04).
After 30 days from the TBI, we observed the main lesion effect on the decrease in
NestinGFP+ cells in the ipsilateral SVZ of the TBI groups compared to the SHAM mice
(p = 0.003, Figure 3G).
These data demonstrate the initial expansion of NestinGFP+ cells in the WT TBI and
KO TBI mice, while a decrease in NSPCs is observed in the KO RUN TBI group with respect
to the KO RUN SHAM mice, which display a powerful increase in SVZ neurogenesis, as
previously shown [28]. Later, the decline in NestinGFP+ cells also becomes evident in the
other TBI groups compared to their SHAM counterparts.
2.4. Inﬂuence of p21 Deletion and Running Session on NSPC Regulation after TBI
The analysis within the injured groups did not reveal at 7 days post TBI any signiﬁcant
differences in the NestinGFP+ sub-populations. In addition, 14 days after TBI, we observe in
the ipsilateral SVZ an increase in the proliferating Nestin GFP+ and in the total proliferation
in the WT RUN TBI, KO TBI and KO RUN TBI group compared to the WT TBI group (WT
TBI vs. WT RUN TBI, p = 0.01, vs. KO TBI and KO RUN TBI p < 0.001; Ki67+ cells: WT
TBI vs. WT RUN TBI, p = 0.01, vs. KO TBI, and KO RUN TBI, p < 0.001, Figure 3F). In
the contralateral SVZ, we observe a strong proliferative response of NestinGFP+ cells in
the KO RUN TBI group, compared to the WT TBI mice (Ki67+ NestinGFP+: KO RUN TBI
vs. WT TBI, p = 0.024, Supplementary Figure S6D), leading to an increase in NestinGFP+
cell pool size (KO RUN TBI vs. KO TBI, p = 0.002, Supplementary Figure S6C), and
consequently in total proliferation (KO RUN TBI vs. WT TBI, p = 0.0048, vs. KO TBI,
p = 0.006, Supplementary Figure S6E). At 30 days post TBI, we did not ﬁnd any difference
within the injured groups in either of the hemispheres.
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These data demonstrate the initial expansion of NestinGFP+ cells in the WT TBI and 
KO TBI mice, while a decrease in NSPCs is observed in the KO RUN TBI group with 
respect to the KO RUN SHAM mice, which display a powerful increase in SVZ 
neurogenesis, as previously shown [28]. Later, the decline in NestinGFP+ cells also 
becomes evident in the other TBI groups compared to their SHAM counterparts. 
 
Figure 3. Time course of NSPCs and type A neuroblast following TBI. (A) Micrographs show the 
decreased proliferation of neural stem/progenitor cells (NSPCs, Ki67+/NestinGFP+ cells) in the KO 
TBI RUN group with respect to the KO TBI mice at 7 days post TBI. (B) Representative pictures 
indicate the significantly decreased number of Ki67+/NestinGFP+ cells in the KO TBI RUN mice in 
comparison to their respective SHAM littermate 14 days after TBI. (C) Histograms show a significant 
increase in the proliferating NSPCs of WT TBI and KO TBI mice in comparison with their respective 
WT SHAM and KO groups (Ki67+ NestinGFP+ cells, ipsilateral: genotype x run x lesion interaction: 
F(1,112) = 11.71, p < 0.001, followed by LSD post-test, WT TBI vs. WT SHAM and KO TBI vs. KO SHAM 
p = 0.015). (D) Graph indicates a significant increase in the total proliferation of WT TBI and KO TBI 
mice with respect to the WT and KO SHAM groups (Ki67+ cells, genotype x run x lesion interaction, 
ipsilateral: F(1,108) = 14.38, p < 0.001, followed by LSD post-test, WT TBI vs. WT SHAM, p = 0.03, KO 
TBI vs. KO SHAM p < 0.001). (C,D) It is also possible to observe the decrease in the NSPCs 
proliferation of KO RUN TBI mice with respect to the KO RUN SHAM group (KO RUN TBI vs. KO 
RUN SHAM, p < 0.001, (C)) and total proliferation (KO RUN TBI vs. KO RUN SHAM, p < 0.001, (D)). 
Figure 3. Time course of NSPCs and type A neuroblast following TBI. (A) Micrographs show the
decreased proliferation of neural stem/progenitor cells (NSPCs, Ki67+/NestinGFP+ cells) in the KO
TBI RUN group with respect to the KO TBI mice at 7 days post TBI. (B) Representative pictures
indicate the signiﬁcantly decreased number of Ki67+/NestinGFP+ cells in the KO TBI RUN mice in
comparison to their respective SHAM littermate 14 days after TBI. (C) Histograms show a signiﬁcant
increase in the proliferating NSPCs of WT TBI and KO TBI mice in comparison with their respective
WT SHAM and KO groups (Ki67+ NestinGFP+ cells, ipsilateral: genotype x run x lesion interaction:
F(1,112) = 11.71, p < 0.001, followed by LSD post-test, WT TBI vs. WT SHAM and KO TBI vs. KO
SHAM p = 0.015). (D) Graph indicates a signiﬁcant increase in the total proliferation of WT TBI and
KO TBI mice with respect to the WT and KO SHAM groups (Ki67+ cells, genotype x run x lesion
interaction, ipsilateral: F(1,108) = 14.38, p < 0.001, followed by LSD post-test, WT TBI vs. WT SHAM,
p = 0.03, KO TBI vs. KO SHAM p < 0.001). (C,D) It is also possible to observe the decrease in the
NSPCs proliferation of KO RUN TBI mice with respect to the KO RUN SHAM group (KO RUN
TBI vs. KO RUN SHAM, p < 0.001, (C)) and total proliferation (KO RUN TBI vs. KO RUN SHAM,
p < 0.001, (D)). (E) Graph displays the reduced proliferation of NSPCs in mice analyzed 14 days after
TBI, when compared to their respective SHAM counterparts (NestinGFP+ cells, ipsilateral: lesion
effect: F(1,105) = 17.21, p < 0.001, $). (F) Histogram shows the decreased total proliferation after 14 days
Int. J. Mol. Sci. 2023, 24, 2911
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after TBI in the WT TBI and KO RUN TBI mice with respect to their SHAM littermates (Ki67+ cells:
genotype x run x lesion interaction: F(1,99) = 17.04, p < 0.001, followed by LSD post-test, WT TBI vs.
WT SHAM, p = 0.029; KO RUN TBI vs. KO RUN SHAM p < 0.001). In the TBI groups, we detected
a signiﬁcant increase in proliferation in the WT RUN TBI mice (F(1,108) = 5.84, p = 0.017, followed
by LSD post-test, WT TBI vs. WT RUN TBI, p = 0.01), as well as an increase in the KO TBI and KO
RUN TBI mice (p < 0.01), in comparison with the WT TBI group. (G) Graph shows the decreased
NSPC population in the ipsilateral SVZ of TBI mice with respect to their SHAM genotypes, 30 days
after the TBI (NestinGFP+ cells: lesion effect: F(1,89) = 9.2, p = 0.003, #). (H) Histogram indicates at
30 days post TBI the increased number of neuroblasts (DCX+ cells) in the TBI group in comparison
with their SHAM littermates (DCX+ cells, ipsilateral, lesion effect: F(1,53) = 20.7, p < 0.001, $). The
comparison within the TBI groups (black histograms) show a signiﬁcant increase in neuroblasts in
the KO RUN TBI mice in comparison with WT TBI, WT RUN and KO TBI mice (genotype x run
interaction: F(1,53) = 7.77, p = 0.007, followed by LSD post-test, KO RUN TBI vs. WT TBI = 0.015,
vs. WT RUN TBI < 0.001, vs. KO TBI = 0.001, a, b, c, respectively). N = 5 mice/group. Statistical
signiﬁcance of LSD post hoc analysis: * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical signiﬁcance
of main lesion effect between SHAM and TBI groups: $ p < 0.001 and # p < 0.01. Multifactorial
analysis with the following three independent variables: genotype, treatment and running, followed
by Fisher’s LSD post hoc tests. Magniﬁcation = 20×. Scale bar = 100 µm. SVZ = subventricular zone.
LV = lateral ventricle.
These data suggest a transitory increase 14 days after the TBI in contralateral sub-
ventricular cell proliferation, which is dependent on physical activity and the lack of the
p21 gene.
2.5. TBI Triggers a Late Increase in Type A Neuroblasts
The analysis of the TBI-induced modulation of type A neuroblasts was carried out by
using the speciﬁc marker DCX. At 7 and 14 days post TBI in the ipsi-lateral region, we did
not detect any signiﬁcant variation in DCX+ neuroblasts in the injured groups compared to
the corresponding SHAM groups, while in the contralateral SVZ, our data showed at 7 days
post TBI a lesion effect on the increase in neuroblasts of the injured groups, compared to
their SHAM counterparts (DCX+ cells: p = 0.004, Supplementary Figure S7A); after 14 days
post TBI, we observed in the contralateral SVZ a lesion effect on the decrease in DCX+
cells of the groups subjected to TBI (p < 0.029, Supplementary Figure S7B). At 30 days post
TBI, we observe a strong increase in both hemispheres in the number of neuroblasts in
the TBI groups, compared to the corresponding SHAM groups (DCX+ cells, ipsilateral:
p < 0.001, Figures 3H and 4A,B, Supplementary Figure S8A–H; contra-lateral: p < 0.001,
Supplementary Figure S7C).
Collectively, these data highlight how the neuroblast population tends to respond late
to TBI, through an increase of the cell number in the ipsilateral SVZ at 30-days following
the trauma, compared to the SHAM groups.
2.6. Inﬂuence of p21 Deletion and Running Session on TBI-Induced Neuroblast Modulation
The analysis between the injured groups at 7 and 14 days post TBI does not show
any TBI-dependent modulation of DCX+ cells in either of the ipsi- and the contralateral
region. At 30 days after the trauma, on the other hand, we ﬁnd an increase in neuroblasts
in the ipsilateral SVZ of the KO RUN TBI group, compared to the other groups (KO
RUN TBI vs. WT TBI = 0.015, vs. WT RUN TBI and KO TBI < 0.001, a, b, c, respectively,
Figures 3H and 4B,C, Supplementary Figure S8B,D,F,H).
Int. J. Mol. Sci. 2023, 24, 2911
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2.6. Influence of p21 Deletion and Running Session on TBI Induced Neuroblast Modulation 
The analysis between the injured groups at 7 and 14 days post TBI does not show any 
TBI-dependent modulation of DCX+ cells in either of the ipsi- and the contralateral region. 
At 30 days after the trauma, on the other hand, we find an increase in neuroblasts in the 
ipsilateral SVZ of the KO RUN TBI group, compared to the other groups (KO RUN TBI 
vs. WT TBI = 0.015, vs. WT RUN TBI and KO TBI < 0.001, a, b, c, respectively, Figures 3H 
and 4B,C, Supplementary Figure S8B,D,F,H). 
 
Figure 4. (A–C). Increase in DCX+ cells in the KO RUN TBI group. Confocal representative micro-
graphs show the increased number of DCX+ cells in the KO RUN TBI mice (C), in comparison with
the KO SHAM (A) and KO TBI (B) mice, 30 days after TBI. Magniﬁcation = 20×. Scale bar = 100 µm.
SVZ = subventricular zone. LV = lateral ventricle.
2.7. TBI Triggers a Boost in Migration toward the Peri-Lesion Cortical Region of KO RUN Mice
The next step was to study the migratory ﬂow of new cells that originated in the SVZ
and were redirected to the damage site in an attempt to contribute to the tissue repair
Int. J. Mol. Sci. 2023, 24, 2911
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process. For this study, in the four groups subjected to trauma, both the NSPCs (Nestin
GFP+) and neuroblasts (DCX+) in their path from the SVZ to the injured cortex were
analyzed. We also analyzed the proliferative fraction of NSPCs and neuroblasts. The data
obtained show that 7 days after TBI in the KO RUN TBI group, there is a signiﬁcant increase
in both NestinGFP+ and Ki67+NestinGFP+ cells within the migration region towards the
injured cortex, compared to the other groups (NestinGFP+: KO RUN TBI vs. WT TBI,
p = 0.017, vs. WT RUN TBI, p = 0.031 and vs. KO TBI p = 0.027, Figure 5A,I,J; Ki67+
NestinGFP+ cells: KO RUN TBI vs. WT TBI, p = 0.027, vs. WT RUN TBI, p = 0.038 and vs.
KO TBI p = 0.049, Figure 5B,I,J).
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Figure 5. Cell migration following TBI. (A) Graph shows the increase in the migratory stream (MS) 
of the KO RUN TBI mice in comparison to the other groups 7 days after the lesion of NestinGFP+ 
cells (genotype x running: F(1.18) = 5.98, p = 0.025, followed by LSD post-test, KO RUN TBI vs. WT 
TBI, p = 0.017, vs. WT RUN TBI, p = 0.031 and vs. KO TBI p = 0.027). (B) Graph shows the increase in 
proliferating NSPCs in the migratory stream (MS) of the KO RUN TBI mice 7 days after the lesion 
(Ki67+ Nestin GFP+ cells: genotype x running: F(1,18) = 5.51, p = 0.03, followed by LSD post-test, KO 
RUN TBI vs. WT TBI, p = 0.027, vs. WT RUN TBI, p = 0.038 and vs. KO TBI p = 0.049). (C) Histograms 
illustrate the enhancement in the MS of KO RUN TBI mice with respect to the other experimental 
condition 14 days after the TBI of NestinGFP+ cells (genotype x run interaction: F(1,18) = 6.55, p = 0.019, 
followed by LSD post-test, KO RUN TBI vs. WT TBI, p < 0.001, vs. WT RUN TBI, p = 0.01 and vs. KO 
TBI, p = 0.023). (D) Graph shows the increment, with respect to the other experimental conditions of 
DCX+ cells, in the MS of KO RUN TBI 14 days after TBI (genotype x run interaction: F(1,18) = 7.07, p = 
0.016, followed by LSD post-test, KO RUN TBI vs. WT RUN TBI, p = 0.024 and vs. KO TBI, p = 0.02). 
(E) Graph shows the increase in the MS of KO RUN TBI mice with respect to the other groups 14 
days after the TBI of proliferating NestinGFP+ (Ki67+/NestinGFP+: genotype x run interaction: F(1,18) 
= 5.07, p = 0.037, followed by LSD post-test, KO RUN TBI vs. WT TBI, p = 0.007, WT RUN TBI, p = 
0.038 and vs. KO TBI, p = 0.0073). (F) Graph shows the increments, with respect to the other 
experimental conditions of proliferating DCX+ cells, in the MS of KO RUN TBI 14 days after TBI 
(Ki67+ DCX+ cells: genotype x run interaction: F(1,18) = 7.07, p = 0.016, followed by LSD post-test, KO 
RUN TBI vs. WT TBI, p = 0.004, WT RUN TBI, p = 0.008 and vs. KO TBI, p = 0.0018). (G) Graph 
indicates an increase in migrating NestinGFP+ cells in the KO TBI and KO TBI RUN mice with 
respect to the WT TBI mice 30 days after TBI (genotype effect: F(1 25) = 3 34 p = 0 011 ^) (H)
Figure 5. Cell migration following TBI. (A) Graph shows the increase in the migratory stream (MS)
of the KO RUN TBI mice in comparison to the other groups 7 days after the lesion of NestinGFP+
cells (genotype x running: F(1.18) = 5.98, p = 0.025, followed by LSD post-test, KO RUN TBI vs. WT
TBI, p = 0.017, vs. WT RUN TBI, p = 0.031 and vs. KO TBI p = 0.027). (B) Graph shows the increase
in proliferating NSPCs in the migratory stream (MS) of the KO RUN TBI mice 7 days after the
lesion (Ki67+ Nestin GFP+ cells: genotype x running: F(1,18) = 5.51, p = 0.03, followed by LSD
post-test, KO RUN TBI vs. WT TBI, p = 0.027, vs. WT RUN TBI, p = 0.038 and vs. KO TBI p = 0.049).
(C) Histograms illustrate the enhancement in the MS of KO RUN TBI mice with respect to the
other experimental condition 14 days after the TBI of NestinGFP+ cells (genotype x run interaction:
F(1,18) = 6.55, p = 0.019, followed by LSD post-test, KO RUN TBI vs. WT TBI, p < 0.001, vs. WT RUN
TBI, p = 0.01 and vs. KO TBI, p = 0.023). (D) Graph shows the increment, with respect to the other
experimental conditions of DCX+ cells, in the MS of KO RUN TBI 14 days after TBI (genotype x
run interaction: F(1,18) = 7.07, p = 0.016, followed by LSD post-test, KO RUN TBI vs. WT RUN TBI,
p = 0.024 and vs. KO TBI, p = 0.02). (E) Graph shows the increase in the MS of KO RUN TBI mice
Int. J. Mol. Sci. 2023, 24, 2911
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with respect to the other groups 14 days after the TBI of proliferating NestinGFP+ (Ki67+/NestinGFP+:
genotype x run interaction: F(1,18) = 5.07, p = 0.037, followed by LSD post-test, KO RUN TBI vs. WT
TBI, p = 0.007, WT RUN TBI, p = 0.038 and vs. KO TBI, p = 0.0073). (F) Graph shows the increments,
with respect to the other experimental conditions of proliferating DCX+ cells, in the MS of KO RUN
TBI 14 days after TBI (Ki67+ DCX+ cells: genotype x run interaction: F(1,18) = 7.07, p = 0.016, followed
by LSD post-test, KO RUN TBI vs. WT TBI, p = 0.004, WT RUN TBI, p = 0.008 and vs. KO TBI,
p = 0.0018). (G) Graph indicates an increase in migrating NestinGFP+ cells in the KO TBI and KO
TBI RUN mice with respect to the WT TBI mice 30 days after TBI (genotype effect: F(1,25) = 3.34,
p = 0.011, ˆ). (H) Histograms show an enhancement of migrating DCX+ cells in the KO TBI and KO
TBI RUN group with respect to the WT TBI mice 30 days after TBI (genotype effect: F(1.25) = 12.64,
p = 0.0015, #). (I,J) Confocal micrographs show the increased density of migrating NestinGFP+ cells
observed in the KO RUN TBI mice with respect to the WT TBI mice, 7 days from TBI. (K,L) Confocal
micrographs illustrate the enhancement of NSPCs (NestinGFP+) and neuroblasts (DCX+) along the
MS of the KO RUN TBI mice compared to the WT TBI group, 14 days post TBI. Arrow indicates
the presence of proliferating NestinGFP+ cells and arrowheads indicate proliferating DCX+ cells.
N = 5 mice/group. Statistical signiﬁcance of LSD post hoc analysis: * p < 0.05, ** p < 0.01 and
*** p < 0.001. Statistical signiﬁcance of main genotype effect between WT and KO groups: # p < 0.01,
ˆ p < 0.05. Two-way ANOVA analysis followed by Fisher’s LSD post hoc tests. Magniﬁcation = 20×.
Scale bar = 100 µm. SVZ = subventricular zone. MS = migratory stream.
In addition, 14 days after the TBI, our data show in the migratory stream (MS) of the
KO RUN TBI group a signiﬁcant increase, compared to the other experimental groups, in
NestinGFP+ cells (KO RUN TBI vs. WT TBI, p < 0.001, vs. WT RUN TBI, p = 0.01 and vs.
KO TBI, p = 0.023, Figure 5C,K,L) and DCX+ cells (KO RUN TBI vs. WT RUN TBI, p = 0.024
and vs. KO TBI, p = 0.02, Figure 5D,K,L). We also observed a marked enhancement in
proliferating migrating cells in the KO RUN TBI group, both in terms of total proliferation
(KO RUN TBI vs. WT TBI, p = 0.046, vs. WT RUN TBI, p = 0.04 and vs. KO TBI, p = 0.0028),
and regarding the proliferative rate of Nestin GFP+ cells (Ki67+ NestinGFP+: KO RUN TBI
vs. WT TBI, p = 0.007, vs. WT RUN TBI, p = 0.038 and vs. KO TBI, p = 0.0073, Figure 5E,K,L),
and neuroblasts (Ki67+ DCX+ cells: KO RUN TBI vs. WT TBI, p = 0.004, vs. WT RUN TBI,
p = 0.008 and vs. KO TBI, p = 0.0018, Figure 5F,K,L).
At 30 days post TBI, we observe a signiﬁcant genotype effect with an increase in the
p21 KO groups in density within the MS of Nestin GFP+ and DCX+ cells, compared to the
WT animals (NestinGFP+: p = 0.011, Figure 5G; DCX+: p = 0.0015, Figure 5H).
Altogether, these data suggest that voluntary physical activity promotes widespread
TBI-dependent migration from SVZ toward the injured cortex of NSCs and neuroblasts in
mice lacking the p21 gene, likely enhancing the neuroprotective and regenerative processes
that occur after the lesion.
2.8. p21 Deletion and Running Session Strongly Inﬂuence the TBI-Induced New-Born Cell
Localization in the Peri-Lesion Cortex
Moreover, we characterized the presence of newborn cells, likely derived from the
SVZ, in the damage site 7, 14 and 30 days after TBI. To this aim, we considered three
different areas of the damage, which were as follows: (i) one area lateral to the lesion
(lateral); (ii) one on the medial side (medial); (iii) the last on the central lower edge of the
lesion (low). In these regions, we counted the total number of NestinGFP+ and DCX+, as
well as the number of NestinGFP+ and DCX+ cells co-expressing BrdU, assuming that most
of these cells that express BrdU might originate in and derive from the SVZ through the
migratory redirection previously analyzed. At 7 days post TBI, we observe a substantial
increase in BrdU+ NestinGFP+ and BrdU+ DCX+ cells in the KO RUN TBI group of animals,
compared to the other experimental conditions both in the lateral region (BrdU+ Nestin
GFP+: KO RUN TBI vs. WT TBI, p = 0.02, vs. WT RUN TBI, p = 0.041 and vs. KO TBI
p = 0.016, Figure 6A,K,L; BrdU+ DCX+: KO RUN TBI vs. WT TBI, p = 0.026, vs. WT RUN
TBI, p = 0.015 and vs. KO TBI p = 0.011, Figure 6B,K,L), as well as in the medial peri-lesion
Int. J. Mol. Sci. 2023, 24, 2911
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site (BrdU+ NestinGFP+: KO RUN TBI vs. WT TBI, p = 0.04, vs. WT RUN TBI, p = 0.027
and vs. KO TBI p = 0.049, Figure 6C; BrdU+ DCX+: KO RUN TBI vs. WT TBI, p = 0.026, vs.
WT RUN TBI, p = 0.015 and vs. KO TBI p = 0.011, Figure 6D).
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 
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Figure 6. Distribution of NSPCs and neuroblasts in the peri-lesion cortex. Seven days post TBI 
(A,C). Graphs show the increased density in the lateral (A) and medial (C) regions that border the 
cortical lesion of the KO RUN TBI mice of Brdu+NestinGFP+ cells (lateral, genotype x running: F(1,24) 
= 4.64, p = 0.041, followed by LSD post-test, KO RUN TBI vs. WT TBI, p = 0.02, vs. WT RUN TBI, p = 
0.041 and vs. KO TBI p = 0.016, (A); medial, genotype X running: F(1,21) = 7.26, p = 0.013, followed by 
LSD post-test, KO RUN TBI vs. WT TBI, p = 0.04, vs. WT RUN TBI, p = 0.027 and vs. KO TBI p = 
0.049, (C)). (B,D) Histograms illustrate in the lateral (B) and medial (D) peri-lesioned cortical area 
of the KO RUN TBI mice the enhanced BrdU+DCX+ cell density (lateral, genotype X running: F(1,21) = 
7.32, p = 0.012, followed by LSD post-test, KO RUN TBI vs. WT TBI, p = 0.026, vs. WT RUN TBI, p = 
0.015 and vs. KO TBI p = 0.011, (B); medial, BrdU+ DCX+: genotype X running: F(1,21) = 7.32, p = 0.012, 
followed by LSD post-test, KO RUN TBI vs. WT TBI, p = 0.026, vs. WT RUN TBI, p = 0.015 and vs. 
KO TBI p = 0.011, (D)). Fourteen days post TBI. (E,F,G) Graphs show, in the medial (E) and low (F) 
peri-injured cortical region of the KO RUN TBI, a significant increase in DCX+ cells (medial, 
Figure 6. Distribution of NSPCs and neuroblasts in the peri-lesion cortex. Seven days post TBI (A,C).
Graphs show the increased density in the lateral (A) and medial (C) regions that border the cortical
lesion of the KO RUN TBI mice of Brdu+NestinGFP+ cells (lateral, genotype x running: F(1,24) = 4.64,
p = 0.041, followed by LSD post-test, KO RUN TBI vs. WT TBI, p = 0.02, vs. WT RUN TBI, p = 0.041
and vs. KO TBI p = 0.016, (A); medial, genotype X running: F(1,21) = 7.26, p = 0.013, followed by LSD
post-test, KO RUN TBI vs. WT TBI, p = 0.04, vs. WT RUN TBI, p = 0.027 and vs. KO TBI p = 0.049, (C)).
(B,D) Histograms illustrate in the lateral (B) and medial (D) peri-lesioned cortical area of the KO
RUN TBI mice the enhanced BrdU+DCX+ cell density (lateral, genotype X running: F(1,21) = 7.32,
Int. J. Mol. Sci. 2023, 24, 2911
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p = 0.012, followed by LSD post-test, KO RUN TBI vs. WT TBI, p = 0.026, vs. WT RUN TBI, p = 0.015
and vs. KO TBI p = 0.011, (B); medial, BrdU+ DCX+: genotype X running: F(1,21) = 7.32, p = 0.012,
followed by LSD post-test, KO RUN TBI vs. WT TBI, p = 0.026, vs. WT RUN TBI, p = 0.015 and
vs. KO TBI p = 0.011, (D)). Fourteen days post TBI. (E,F,G) Graphs show, in the medial (E) and
low (F) peri-injured cortical region of the KO RUN TBI, a signiﬁcant increase in DCX+ cells (medial,
genotype x run interaction: F(1,20) = 14.14, p = 0.0012, followed by LSD post-test, KO RUN TBI vs. WT
RUN TBI, p = 0.007 and vs. KO TBI, p = 0.03, (E) and low (genotype x run interaction: F(1,20) = 7.52,
p = 0.012, followed by LSD post-test, KO RUN TBI vs. WT TBI and WT RUN TBI, p < 0.001, vs.
KO TBI, p = 0.03, (F)), as well as an increase in BrdU+DCX+ cells in the low side ((G), genotype x
run interaction: F(1,20) = 7.52, p = 0.012, followed by LSD post-test, KO RUN TBI vs. WT TBI and
WT RUN TBI, p < 0.001, vs. KO TBI, p = 0.03). Thirty days post TBI. (H,I) Histograms illustrate
the signiﬁcant increment in the KO RUN TBI group of NestinGFP+ cells in the medial (H) and low
(I) regions (medial: genotype x run interaction: F(1,15) = 8.26, p = 0.011, followed by LSD post-test,
KO RUN TBI vs. WT TBI, p < 0.001, vs. WT RUN TBI, p = 0.008, vs. KO TBI, p = 0.01, (H); low:
genotype x run interaction: F(1,15) = 6.71, p = 0.02, followed by LSD post-test, KO RUN TBI vs. WT
TBI, p < 0.001, vs. WT RUN TBI and KO TBI p = 0.01, (I)). (J) Graph shows the increase in DCX+ cells
in the lateral cortical regions of KO RUN TBI mice, 30 days after TBI (genotype x run interaction:
F(1,15) = 6.51, p = 0.022, followed by LSD post-test, KO RUN TBI vs. WT TBI, p = 0.015, vs. WT RUN
TBI, and KO TBI, p = 0.047). (K,L) Confocal representative pictures show the speciﬁc localization of
NestinGFP+ (arrows) and DCX+ (arrowheads) cells in the lateral cortical region lining the lesion of the
KO RUN TBI mice, at 7 days post TBI (K). At the same time-point in the WT TBI group, we observe
only a limited number of NestinGFP+ (arrow) cells in the peri-lesioned lateral side. (M,N) Confocal
representative micrographs show that after 14 days from TBI, it is possible to observe in the medial
side of the lesion of KO RUN TBI mice (M) a high density of NestinGFP+ (green), and DCX+ (blue)
cells co-localizing with BrdU (red). In the same area of the WT TBI (N) mice, we detect a much lower
density of cells. (O,P) The confocal pictures show the accumulation of DCX+ and BrdU+DCX+ cells
in the low cortical peri-lesion area of the KO RUN TBI mice (P), which is not detectable in the WT TBI
group (O). N = 5 mice/group. Statistical signiﬁcance: * p < 0.05, ** p < 0.01 and *** p < 0.001. Two-way
ANOVA analysis and Fisher’s LSD post hoc tests. Magniﬁcation = 20×. Scale bar = 100 µm.
We also observed a genotype effect on the number of total NestinGFP+ and DCX+ cells
in the lateral region (NestinGFP+ cells: p < 0.001, Supplementary Figure S9A; DCX+ cells:
p = 0.02, Supplementary Figure S9B) and in the medial region (NestinGFP+ cells: p < 0.001,
Supplementary Figure S9C; DCX+ cells: p < 0.001, Supplementary Figure S9D), suggesting
that deletions of the p21 gene may play an important role in increasing the number of new
neurons in the injured cortical regions.
At 14 days post TBI, a signiﬁcant effect in the lateral peri-lesion region of p21 dele-
tion and running on the increase in NestinGFP+ cells (genotype effect: p = 0.0013; run
effect: p = 0.0038, Supplementary Figure S9E) and BrdU+ NestinGFP+ cells (genotype effect:
F(1,20) = 9.65, p = 0.0056; run effect: F(1.20) = 4.64, p = 0.043, Supplementary Figure S9F)
occurs. In the medial and low region of the lesion, we observed a signiﬁcant increase in
the DCX+ populations in the KO RUN TBI group (medial: KO RUN TBI vs. WT RUN TBI,
p = 0.007 and vs. KO TBI, p = 0.03, Figure 6E,M,N; low: KO RUN TBI vs. WT TBI, WT RUN
TBI and KO TBI, p < 0.001, Figure 6F) and in BrdU+ DCX+ limited to the low zone (KO
RUN TBI vs. WT TBI and WT RUN TBI, p < 0.001, vs. KO TBI, p = 0.03, Figure 6G).
At 30 days post TBI, a signiﬁcant effect of genotype on the increase in NestinGFP+
cells can be observed in the lateral peri-lesion region (lateral: genotype effect: p < 0.001).
Moreover, we detect a signiﬁcant increase in NestinGFP+ cells in the KO RUN TBI group
when compared to the other conditions in the medial and low cerebral region (medial:
KO RUN TBI vs. WT TBI, p < 0.001, vs. WT RUN TBI, p = 0.008, vs. KO TBI, p = 0.01,
Figure 6H; low: KO RUN TBI vs. WT TBI, p < 0.001, vs. WT RUN TBI and KO TBI p = 0.01,
Figure 6I,O,P). Finally, in the lateral region, we observed a strong increase in DCX+ cells in
the KO RUN TBI animals, compared to the other three injured groups (KO RUN TBI vs.
WT TBI, p = 0.015, vs. WT RUN TBI, and KO TBI, p = 0.047, Figure 6J,O,P), as well as in
Int. J. Mol. Sci. 2023, 24, 2911
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BrdU+ neuroblasts (DCX+ BrdU+ cells: KO RUN TBI vs. WT TBI, p = 0.013, vs. WT RUN
TBI, p = 0.047 and vs. KO TBI p = 0.05).
Taken together, these data demonstrate how TBI produces a signiﬁcant migratory ﬂow
of stem cells and neuroblasts towards the injured region in the KO RUN TBI group, with
the consequent accumulation of these populations in the cortical area closely adjacent to
the lesion.
2.9. Volume of the Lesion Is Not Affected by p21 Deletion or Running
Subsequently, we wanted to evaluate whether the cellular dynamics of proliferation,
differentiation and especially migration that occur after TBI could modulate the macro-
scopic neuroanatomical recovery in our experimental groups. To this end, we measured
the volume of the lesions in the cerebral cortex directly affected by the lesion of the four
groups at 14 and 30 days post lesion, by the application of Cavalieri’s estimator of morpho-
metric volume. Our analysis does not evidence any signiﬁcant difference within the four
experimental conditions (Figure 7A,B). Moreover, the data related to the lesion’s variation
over time evidence the signiﬁcant effect of time in contributing to lesion volume reduction
(time effect: p < 0.05, Figure 7C), conﬁrming that after TBI, the brain activates mechanisms
that promote the processes of regeneration and anatomical recovery.
2.10. Running and p21 Deletion Induce Partial Post-TBI Functional Recovery
Since the controlled cortical impact (CCI) procedure was conducted in the cortical
area corresponding to the primary motor cortex that controls the right forelimb, the next
step was the evaluation of the putative relevance of injury-induced SVZ neurogenesis
in ameliorating the functional recovery after TBI. To this aim, we used the Ladder Rung
Walking test as a behavioral task to assess the skilled walking and right forelimb stepping,
placing and coordination of the animals by measuring the number of mistakes in foot
placement of the right forelimb during a 50 cm walk. We carried out this analysis at
different time points, including 1 day before TBI (pre-TBI) to determine the functional
baseline of the mice and 2, 7, 14 and 30 days after TBI. In the statistical analysis (ANOVA
analysis for repeated measures), we considered the following four independent variables
and their interaction: (1) the genotype of the animals (p21 WT or KO), (2) the surgical
procedure (TBI or SHAM), (3) the physical activity (running or sedentary) and (4) the effect
of the period of training (time). First of all, the data reveal the functional recovery over time,
as demonstrated by the decrease in mistakes in the different TBI groups through the time
points and by the statistical analysis of the time variable (time effect: p < 0.05, Figure 7D).
Moreover, even if the statistical analysis of the independent variables genotype and running
was not signiﬁcant, the study of the interaction among genotype, physical activity and
treatment (TBI or SHAM) was signiﬁcant in inﬂuencing the functional outcome (genotype
x surgery x running interaction, p < 0.05, Figure 7D). We did not ﬁnd any signiﬁcant
differences among the SHAM groups that maintained a similar number of errors at every
time point considered. Moreover, at P2, we noticed an increased number of errors in the
four TBI groups if compared to SHAM animals, an effect that seems to be more prominent
in WT RUN animals. This fact conﬁrms that the surgical procedure by itself does not
provoke any impairment in the functional performance of the animals and that contusion
is a key factor for the higher number of mistakes committed by animals (TBI vs. SHAM,
p < 0.001, Figure 7D). The graph even shows that at P7, the KO RUN TBI mice display
better functional performances after the trauma than the other TBI groups (P7: KO RUN
TBI vs. WT TBI, WT RUN TBI and KO TBI, p < 0.001). Moreover, we observe that only
in KO RUN TBI mice, the number of errors is not signiﬁcantly different compared to the
SHAM groups at 7, 14 and 30 days after TBI (P7, P14, P33: p > 0.05 KO RUN TBI vs. all
SHAM groups, Figure 7D). The area under the curve analysis conﬁrms the TBI-dependent
functional deterioration (TBI vs. SHAM, p < 0.001), while within the TBI groups, we
observe a signiﬁcant decrease in errors in the KO RUN TBI group compared to the other
experimental groups (KO RUN TBI vs. WT TBI, p < 0.05, vs. WT RUN TBI and KO TBI,
Int. J. Mol. Sci. 2023, 24, 2911
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p < 0.001, Figure 7E). As a whole, our data suggest that the addictive effect of running and
p21 deletion could be effective in promoting a partially higher functional outcome after TBI.
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Figure 7. Anatomical and functional recovery following TBI. (A,B) Graphs show the average 
volumes of the lesions 14 (A) and 30 (B) days after the TBI procedure in the four experimental 
groups. (C) Histograms indicate the decreased average volumes of the lesions from 14 to 30 days 
post TBI in the four different experimental conditions (time effect: F(4,28) = 22.08; p < 0.05). (D) The 
graph indicates the mean number of errors per group at each time point. The same color shows an 
Figure 7. Anatomical and functional recovery following TBI. (A,B) Graphs show the average volumes of
the lesions 14 (A) and 30 (B) days after the TBI procedure in the four experimental groups. (C) Histograms
Int. J. Mol. Sci. 2023, 24, 2911
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indicate the decreased average volumes of the lesions from 14 to 30 days post TBI in the four different
experimental conditions (time effect: F(4,28) = 22.08; p < 0.05). (D) The graph indicates the mean
number of errors per group at each time point. The same color shows an experimental condition and
its relative SHAM control. The statistical analysis evidenced signiﬁcant effects of the time variable
(time effect F = 9.949 p < 0.05, Figure 7D). Within the TBI groups, we found that at 7 days post TBI,
the KO RUN TBI animals demonstrated better performances with respect to the other conditions and,
notably, a number of mistakes comparable to their SHAM control time effect (P7: genotype x run
interaction: F(1,64) = 7.65 p = 0.032, followed by LSD post-test, KO RUN TBI vs. WT TBI, WT RUN
TBI and KO TBI, p < 0.001). For the statistical analyses of the volumes of the lesions, we performed a
multifactorial analysis with the following three independent variables: genotype; running and time.
(E) The histograms represent the area under the curve analysis and show the increase in errors in
the TBI groups (lesion effect F(1,48) = 153; p < 0.001, $); moreover, in the TBI groups, the statistical
analysis indicates a decrease in errors in the KO RUN TBI mice in comparison with the other TBI
groups (genotype x run x lesion interaction, F(1,48) = 12.92, p < 0.001, followed by LSD post-test, KO
RUN TBI vs. WT TBI, p = 0.011, vs. WT RUN TBI, and KO TBI, p < 0.001). The behavioral data of
the Ladder Rung Walking task have been analyzed by a multifactorial analysis with the following
four independent variables: genotype, running, time and the treatment. The area under the curve
statistics were evaluated by multifactorial analysis with the following three independent variables:
genotype, treatment and running. (N = 8 mice/group). The post hoc analyses have been conducted
via by Fisher’s LSD post hoc tests. Statistical signiﬁcance: * p < 0.05 and *** p < 0.001.
3. Discussion
Several studies have suggested that NSCs may retain or even potentiate their self-
renew ability following injury, in order to produce additional neural progenitors and
neuroblasts, which migrate toward the damaged tissue and contribute to the post-traumatic
neuro-regenerative processes [32,33]. If this hypothesis is conﬁrmed, new strategies to
enhance neurogenesis could be very useful to increase the number of new neurons that can
beneﬁt the cortical region that is directly involved in the damage. In this study, we have
demonstrated that the concomitant deletion of p21 and physical activity play a powerful
role in enhancing the subventricular neurogenic post-traumatic response and improving
functional recovery.
From the comparison between the TBI and SHAM groups, we can observe a clear
dynamic in the post-traumatic SVZ neurogenic response, which results in the early activa-
tion of type B cells, followed over time by a net decrease in the Nestin GFP+ population
and a concomitant increase in type A neuroblasts, leading to a signiﬁcant increase in this
population at 30 days post TBI. From such evidence, we can hypothesize that in our ex-
perimental model, TBI triggers a highly speciﬁc pro-neurogenic process within the SVZ,
characterized by temporally different neurogenic responses within the sub-populations
considered. These data are in apparent contradiction with a study that demonstrates that
transit-amplifying cells (type C cells) are the main cell type responsible for the injury-
induced increase in cell proliferation, with no contribution from either GFAP+ or DCX+
cells [34]. However, in a recent work, a gradual increase over time in proliferation (from
day 1 to day 7 post TBI) associated with an increase in NestinGFP+/GFAP+ NSCs and
DCX progenitors within the SVZ of rats has been observed [21]. In another single-cell
transcriptomics study, it has been demonstrated that brain injury is able to transform
dormant NSCs into primed quiescent and active NSCs, with the concomitant activation of
protein synthesis and cell cycle genes [22]. From these and other studies clearly emerges
a strong discrepancy in the post-traumatic SVZ neurogenic response, derived from the
high heterogeneity of the experimental protocols used, differing signiﬁcantly from each
other in the type of damage applied, in the proliferative/differentiative markers used and
in the length of post-traumatic time-points, as well as by the utilization of different mice
strains [35].
The comparison of the post-traumatic neurogenic response within TBI groups clearly
demonstrates that the p21 deletion and pre-traumatic voluntary physical activity over a
Int. J. Mol. Sci. 2023, 24, 2911
17 of 24
12-day period exert a potent proneurogenic effect, which belatedly results in a signiﬁcant
increase in the population of DCX+ neuroblasts at 30 days post TBI. This stimulating effect of
post-TBI neurogenesis observed in the KO RUN TBI mice differs from what was observed in
the KO RUN SHAM group, in which we detected a considerable increase in subventricular
proliferation, which was signiﬁcantly higher than in the KO RUN TBI group 7 and 14 days
after injury. In this regard, we might speculate that the strong proliferative increase in the
KO RUN mice at 7 and 14 days post SHAM could be the result of the hyper-proliferation of
running-activated NSCs as previously observed [28], an event that was not observed in the
KO RUN TBI group, which displayed a late pro-neurogenic response. These differences
show how the modulation of the sub-populations of newborn cells within the SVZ is very
diversiﬁed, depending on the external stimuli, and furthermore lead us to hypothesize that
in the p21 ko mice, the temporally consequential effects of physical activity and trauma
establish a well-deﬁned series of cellular and microenvironment modiﬁcations that, on the
one hand, require well-deﬁned timing to optimize their proneurogenic effect whereas on
the other hand, the differ profoundly from the effects observed if these external stimuli
are provided separately. Alternatively, we can hypothesize that a third pro-neurogenic
stimulation, the TBI, to the SVZ neurogenic niche of the KO RUN SHAM mice could
lead to an excessive overload in the proliferative rate, with the consequent depletion of
the NSC/progenitor pool, as previously observed following a prolonged running period
(21 days) in p21 ko mice [28]. Furthermore, our study examined the effects of p21 deletion
and running on the migration of newborn neurons after cortical brain injury. In the non-
injured SVZ, post-mitotic neuroblasts move from SVZ to the olfactory bulb along the rostral
migratory stream (RMS) [36]. When cortical regions are injured, neural progenitors start
to migrate outside the RMS into the adjacent tissues, including the corpus callosum (CC)
and the peri-lesioned cortex [35,37]. In this context, interesting evidence that arose from
our study is represented by the observation of the widespread migration of NestinGFP+
and DCX+ cells from the SVZ towards the damaged peri-cortical regions in the KO RUN
TBI group. A very intriguing aspect to take into consideration is the high fraction of
proliferation observed in the migrating cells, testifying the presence of cytogenesis within
the migratory ﬂow, far from the SVZ neurogenic niche. The cytogenesis observed within
the migratory ﬂow both in the NestinGFP+, and to a lesser extent in the DCX+ cells, could
represent an additional neurogenic response that is able to further increase the pool of NSCs
and progenitor cells involved in the unknown tissue repair processes and post-traumatic
functional improvement. Both processes are observed in all the groups subjected to TBI,
although the deletion of p21 associated with physical activity greatly increases its extent.
This evidence conﬁrms what was observed in a previous study, indicating a dramatic
increase in the migration of post-mitotic neuroblasts along the RMS in p21 ko mice after a 5-
and 12-day running session [28]. Moreover, the widespread migration of NestinGFP+ cells
from the SVZ towards the injured cortex could account for the transient decrease in the
NestinGFP+ population observed within the SVZ at 14 days post TBI; in this case, a process
of cellular evasion could be outlined in the phases immediately following the trauma,
which leads to the gradual impoverishment of the NestinGFP+ population within the SVZ.
An explanation for the increased cell migration in the KO RUN TBI mice is still required and
further studies will evaluate the impact of p21 deletion and running on the main pathways
that regulate neuroblast migration. In this regard, several groups have demonstrated the
involvement of trophic factors and their receptors in the microenvironment that promotes
neuroblast migration in both the naïve and post-lesioned brain, including stromal cell-
derived factor 1 (SDF)/C-X-C motif chemokine 4 (CXCR4), brain-derived neurotrophic
factor (BDNF)/tropomyiosin receptor kinase B (TrkB) and vascular endothelial growth
factors (VEGF)/VEGF receptor [37–39]. In particular, it was found that BDNF caused SVZ
cells to emigrate toward cerebral regions [40,41] in a concentrated manner [42]; and also
that either pre- or post-traumatic exercise increased cerebral BDNF protein expression as
compared to non-exercised animals [43–46], suggesting a putative role of BDNF in the
increase in migration in pre-exercised animal trauma.
Int. J. Mol. Sci. 2023, 24, 2911
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In our study, the large migratory ﬂow of NSCs/neuroblasts observed in the KO
RUN TBI group translates into a signiﬁcant accumulation of newly generated neurons
in the tissue bordering the cortical lesion. Using a long-term BrdU assay, we have also
demonstrated that a large fraction of these neuroblasts are newly born and are most
likely of subventricular origin. We do not know exactly the differentiative fate of these
cells nor their functional role within the lesioned microenvironment, because migrating
NSPCs take 1 to 3 months to fully maturate into neurons [47]; furthermore, in our study,
we detected only a minimal fraction of cells that co-expressed the markers BrdU and
NeuN (marker of mature neurons) at 30 days post injury, in full agreement with previous
works [48,49], demonstrating the nearby absence of newly born mature neurons in peri-
lesion regions. The formation of newly generated mature neurons in the cortex following
injury is controversial. Recent works suggest that the cortex microenvironment may
favor glial differentiation, resulting in the downregulation of DCX expression and the
concomitant cortical up-regulation of Olig2 [37,50] and of Shh [51], which trigger glial
differentiation [35]. Pous et al. identiﬁed the ﬁbrinogen released in the microenvironment
SVZ by the leaky vasculature after injury, a key factor driving the differentiation of NSPCs
into astrocytes via the activation of the BMP signaling pathway [52]. Whatever the fate of
the NSPCs of p21 runner mice after TBI, we hypothesize that their considerable increase in
the peri-lesioned cortical region could be beneﬁcial for the neuro-reparative response, as
evidenced by the improvement of the Ladder Walking Test observed in the KO RUN TBI
mice. In agreement with our hypothesis, Dixon et al. demonstrated that selective NSPC
ablation induces a reduction in the number of neuroblasts migrating toward the injury
with the consequent decrease in residential neuron and glial cells in the peri-lesion cortex
and reduced locomotor recovery [53]. We believe that a strong increase in the number
of NSPCs in the peri-injury cortical region of KO RUN TBI mice could greatly enhance
the tissue stabilization processes of the injury milieu, allowing neuroprotection through
the increased inﬂux of neuro-protective factors, such as BDNF and VEGF, which can in
turn promote neuronal survival, local glial proliferation, reduced gliosis and functional
recovery. Our study is descriptive and one of the main limitations of this study is the lack
of analysis of molecular mechanisms that can partially explain the results obtained. In
this regard, however, some of our preliminary evaluations have highlighted the speciﬁc
role of p21 deletion and physical activity in the subventricular neurogenic niche capable of
downregulating the expression of anti-neurogenic genes in the BMP2 pathway. This event
could trigger NSCs to exit from the quiescent state, allowing them to differentiate into the
neuronal lineage [54].
4. Material and Methods
4.1. Animals
Male wild-type and p21-null mice [55] of the same genetic background (129Sv/c57BL6;
50:50; https://www.jax.org/strain/003263, accessed on 12 February 2015) were housed
under a continuous 12 h light/12 h dark cycle at a constant temperature of 21 ◦C, with
complete availability of water and food. Nestin green ﬂuorescent protein mice (C57BL/6
background; kindly provided by Dr. G. Enikolopov) express GFP driven by the Nestin
promoter [56]. Nestin-GFP mice were crossed with WT and knockout mice to obtain WT
and KO/NestinGFP+ mice, which were interbred at least four times before further analysis,
generating the different genotypes under study.
All experiments were performed blind for the different experimental conditions. We
analyzed 5 mice per group in the immunohistochemistry study and 8 mice group in the
functional analysis.
4.2. Running Paradigm and BrdU Administration
Mice subjected to the 12-day running session were housed in a running cage (2 mice
per cage) and their running activity was measured with a speedometer. Moreover, the mice
had been treated with BrdU administered in their drinking water (B5002, Sig-ma; 0.5 g/L)
Int. J. Mol. Sci. 2023, 24, 2911
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from day 7 until the end of the running session, in order to label proliferating NPCs and
neuroblasts and to follow the fate of their progeny. Depending on their genotype (WT
or p21 KO) and on their surgical procedure (SHAM or TBI), the mice were subdivided
according to the following sedentary or running protocols: WT sedentary (WT SHAM),
WT running (WT RUN SHAM), p21 ko sedentary (KO SHAM) and ko running (KO RUN
SHAM), and WT sedentary (WT TBI), WT running (WT RUN TBI), p21 ko sedentary (KO
TBI) and p21 ko running (KO RUN TBI).
4.3. Controlled Cortical Impact (CCI) Injury
Mice were anesthetized with isoﬂurane and positioned within a mouse stereotaxic
frame. Following a longitudinal skin incision, a 3 mm diameter craniotomy was per-
formed at the following stereotaxic coordinates: antero-posterior (AP): +0.5 mm; lateral
−0.5 mm [57]. Traumatic brain injury was performed at the cortical level with a ﬂat, 3 mm
diameter metal tip attached to the CCI device (PinPoint Precision Impactor, Stoelting, Wood
Dale, IL, USA), at an impact speed of 3 m/sec, time of impact of 150 ms and a depth of 2 mm
below the dura, corresponding to the cerebral region of the primary cortex, which controls
the ﬁne movements of the right forelimb (TBI groups). After the impact, the animals were
sutured with absorbable suture thread, housed in their home cage and put on a heated plate
for 3/4 h in order to control their body temperature during their recovery from anesthesia.
Animals were treated following the Italian Ministry of Health and directive 2010/63/EU
guideline nr 785/19 PR. Animals subjected to the surgical procedures described above
without cortical impact represented the SHAM groups.
4.4. Experimental Procedures
First, 12–14-week-old male p21 wt and knockout mice ran for 12 days in free running
wheels; from 7 to 12 days of running, each group of animals received BrdU dissolved in
drinking water, to mark the cells that underwent DNA replication and to follow the fate of
their progeny. On the twelfth day of running, the mice were subjected to the controlled
cortical impact (CCI) surgical procedure as described before.
The mice were sacriﬁced at the following three different time points: seven (P7),
fourteen (P14) and thirty (P30) days after the CCI procedure (Figure 1A). By immunohis-
tochemical assays, we analyzed the following three different regions of the injured brain:
(i) the ipsi- and contralateral SVZ to the lesion to evaluate the post-traumatic neurogenic
response; (ii) the migratory route of new-born cells originating in the SVZ that were re-
directed toward the injured cortical regions; (iii) 3 different cortical sites (medial, low
and lateral) of the lesion to detect the new-born progenitors that reached the injured area
and that are supposed to contribute to ameliorating the outcome after TBI (Figure 1B). To
evaluate the possible functional recovery of the mice subjected to TBI, we performed the
Ladder Rung Walking Test at 2, 7, 14 and 30 days post TBI (Figure 1A).
4.5. Immunohistochemistry
At 7, 14 and 30 days post TBI, the animals were sacriﬁced by trans-cardiac perfusion
with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS); the brains were
collected and kept overnight at −4 ◦C in PFA. They were subsequently equilibrated in
sucrose diluted at 30% and ﬁnally cryopreserved at −80 ◦C. Slicing was carried out by
embedding the brain in Tissue-Tek OCT (Sakura, Torrence, CA, USA) and then cut using a
cryostat at −25 ◦C throughout the whole rostro-caudal extent. The coronal sections were
processed in a one-in-six series protocol at a 40 µm thickness. Sections were then stained for
multiple labelling using different ﬂuorescence techniques. Sections were initially washed
with 0.1 M glycine for 10 min, followed by permeabilization using 0.3% Triton X-100 in
PBS for another 10 min. The sections were then incubated for 30 min in a blocking solution
that contained 3% normal donkey serum (NDS) in 0.3% Triton X-100 in PBS to saturate
the speciﬁc sites, followed by incubation with the same blocking solution that contained
primary antibodies for 16–18 h at 4 ◦C. The primary antibodies used were goat polyclonal
Int. J. Mol. Sci. 2023, 24, 2911
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antibodies, which were used against DCX (Santa Cruz Biotechnology, Dallas, TX, USA; Cat#
Sc-8066; 1:300); a rabbit monoclonal antibody was used against Ki67 (Lab Vision, South
San Francisco, CA, USA, Cat# RM-9106-S; 1:150), whereas a mouse monoclonal antibody
was used against GFAP (Sigma, St. Louis, MO, USA, Cat# G6171; 1:500). The detection
of BrdU-positive cells consisted of denaturing DNA with 2N HCl for 45 min at 37 ◦C to
facilitate antibody access. The sections were then incubated with 0.1 M sodium borate
buffer at pH 8.5, followed by overnight incubation at 4 ◦C with a rat anti-BrdU primary
antibody (Abcam, Cambridge, UK, Cat# ab6326; 1:300) diluted in TBS that contained
0.1% Triton, 0.1% Tween, and 3% normal donkey serum (blocking solution). To observe
primary antibody binding, donkey secondary antibodies against rat (BrdU) and rabbit
(Ki67) conjugated to Cy3 (Jackson ImmunoResearch, West Grove, PA, USA; 1:200 in PBS),
and against goat (DCX) and mouse (GFAP) antibodies conjugated to Alexa-647 (Invitrogen,
San Diego, CA, USA; 1:300 in PBS) were used. Nuclei were observed by incubating sections
with Hoechst (1:500).
4.6. Cell Counting
Cell numbers in the SVZ were obtained with stereological analysis, by counting the
cells that expressed the indicated markers and were visualized with confocal microscopy
throughout the whole rostro-caudal extent of the SVZ in a one-in-ten series of 40 µm
free-ﬂoating serial coronal sections (240 µm apart). The cell numbers obtained for each
SVZ section were divided for the corresponding area of the section to obtain the average
number of SVZ cells per 100 mm2. The areas were obtained by tracing the outline of the
whole SVZ bulb, identiﬁed by the presence of cell nuclei stained by Hoechst on a digital
picture captured and measured using ImageJ software (Version 1.52t released 30 January
2020) [58]. A CellSens Standard system (OLYMPUS) was used to record z-stack images,
and thus conﬁrm the colocalization of multiple labeled cells in the SVZ. To assess the neural
stem/progenitor cell and neuroblast cell numbers in the migratory stream and perilesional
region, non-biased cell number estimations were performed on the 5 most central rostro-
caudal sections around the injury epicenter (as determined using cresyl violet-stained
sections), which were 30 µm thick and 180 µm apart. The count of the labelled cells in the
regions bordering the lesion was carried out within a frame of 300,000 mm2.
4.7. Estimation of Lesion Volume
On a Polysine microscope slide (Thermo Scientiﬁc, Waltham, MA, USA), a number
of brain slices that permitted us to comprehend the entire damage extension were placed.
Then, images of the sections were acquired by ﬂuorescent microscopy at a magniﬁcation
of 4×. The areas of the damage (mm2) on each section were estimated via the “Poly-
gon Selection” tool of ImageJ and following this the total volume of brain damage (mm)
was calculated by Cavalieri’s estimator of morphometric volume, which is as follows:
VC = d (Σ yi) − (t) Ymax, where d is the distance between the sections contained in a well
(d = 240 µm), yi is the area of a single section, t is the section thickness (t = 40 µm) and
yMAX is the maximum value of y. The factor (t) yMAX is subtracted from the basic equation
as a correction for overprojection.
4.8. Ladder Rung Walking Task
The apparatus is made by two see-through walls of Plexiglass of 1 m of length and 20
cm of height with removable metal rungs (3 mm of diameter) at a minimum distance of
1 cm. The ladder is placed at a minimum of 50 cm above the ground with a neutral cage
on one side, from which the mice start the task, and the home cage with the littermates at
the other. The width of the apparatus can be adjusted in order to prevent the animals from
turning around. Animals were tested with a regularly 2 cm interspaced rung pattern one
day before the CCI procedure (pre TBI) to determine the baseline scores and 2 (P2), 7 (P7),
14 (P14) and 30 (P30) days after the surgery to evaluate the functional recovery. Every
mouse underwent 4 trials with a ladder length of 50 cm during which they were recorded
Int. J. Mol. Sci. 2023, 24, 2911
21 of 24
with a camera placed at one side of the apparatus in order to obtain a clear view of the
considered limb (right forelimb). The scoring was carried out through the observation of the
recordings in slow-motion and counting the number of errors for each trial (foot placement
accuracy analysis). The following errors were considered: total miss (the limb completely
missed a rung), deep slip (the limb initially reached the rung but then slipped off, causing
a fall when weight-bearing) and slight slip (the limb slipped off when weight-bearing but
not causing a fall or an interruption of the gait). The mean number of errors per trial was
then standardized for a ladder length of 1 m. The areas under the curve were calculated
with Prism 5.
4.9. Statistical Analysis
The data on the cellular responses after TBI in the SVZ have been analyzed through a
three-way ANOVA, with genotype, running and treatment as the independent variables.
Cell migration and number of cells in the peri-lesioned area have been analyzed through
a two-way ANOVA, with genotype and running as the independent variables. Lesion
volumes at different time-points have been analyzed through a mixed ANOVA, with
genotype, running and time as the independent variables. Finally, the behavioral data
from the Ladder Rung Walking task have been analyzed through a mixed ANOVA, with
genotype, running, time and treatment as the independent variables. Fisher’s LSD post
hoc tests have been conducted whenever necessary. Analyses have been performed with
GraphPad Prism 5 software for the two-way ANOVA and with Statistica software 14.0
(Dell Software) for the three-way and mixed ANOVA studies.
5. Conclusions
The data obtained in this study reveal how the interaction between physical activity
and p21 gene deﬁciency plays an important role in neurogenic and migratory mechanisms
in response to traumatic injury. Our data do not reveal whether the increase in neuroblasts
within the SVZ and their subsequent migration towards the lesioned cortex is a process
capable of increasing the rate of functionally active newly mature neurons. However, the
data obtained in the Ladder Rung Walking Test represented an indirect clue relating the
correlation within the KO RUN TBI group between the cellular processes that take place in
the post-traumatic sequelae and an improvement in the functional response underlying
that particular task. We believe that this study can offer interesting perspectives for future
pre-clinical strategies aimed at investigating the role of physical activity and NSCs in the
post-traumatic neuro-regenerative response.
Supplementary Materials: The supporting information can be downloaded at: https://www.mdpi.
com/article/10.3390/ijms24032911/s1.
Author Contributions: Conceptualization, S.F.V.; methodology, J.I.B., V.M., V.N.d.R., S.M., S.F.V.;
software, V.M., D.S.; validation, S.F.V.; formal analysis, J.I.B., V.M., V.N.d.R., S.M., S.F.V.; investigation,
J.I.B., V.M., V.N.d.R., D.S., S.M., S.F.V.; resources, S.F.V.; data curation, J.I.B., S.F.V.; writing—original
draft preparation, J.I.B., S.F.V.; writing—review and editing, S.F.V., J.I.B., V.M.; visualization S.F.V.;
supervision, S.F.V.; funding acquisition, S.F.V. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by Filas Regione Lazio: GAEPFARIOLI; IBCN/CNR Starting
Grant Fund: GAEPFARIOLI.
Institutional Review Board Statement: The animal study protocol was approved by the Ethics
Committee of Ministry of Health (785/PR, 29/11/2019).
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conﬂicts of Interest: The authors declare no conﬂict of interest.
Int. J. Mol. Sci. 2023, 24, 2911
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Role of Running-Activated Neural Stem Cells in the Anatomical and Functional Recovery after Traumatic Brain Injury in p21 Knock-Out Mice.
Battistini, Jonathan Isacco,Mastrorilli, Valentina,Nicolis di Robilant, Vittoria,Saraulli, Daniele,Marinelli, Sara,Farioli Vecchioli, Stefano