Iannucci et al. Journal of Neuroinflammation
https://doi.org/10.1186/s12974-024-03146-z(2024) 21:165

Journal of Neuroinflammation

Open Access

RESEARCH

Traumatic brain injury alters the effects
of class II invariant peptide (CLIP) antagonism
on chronic meningeal CLIP + B cells,
neuropathology, and neurobehavioral
impairment in 5xFAD mice
Jaclyn Iannucci1, Reagan Dominy1, Shreya Bandopadhyay1, E. Madison Arthur1, Brenda Noarbe3,
Amandine Jullienne3, Margret Krkasharyan3, Richard P. Tobin4, Aleksandr Pereverzev1, Samantha Beevers1,
Lavanya Venkatasamy1, Karienn A. Souza1, Daniel C. Jupiter5, Alan Dabney6, Andre Obenaus3,
M. Karen Newell-Rogers1,2* and Lee A. Shapiro1*
Abstract
Background Traumatic brain injury (TBI) is a significant risk factor for Alzheimer’s disease (AD), and accumulating
evidence supports a role for adaptive immune B and T cells in both TBI and AD pathogenesis. We previously identified
B cell and major histocompatibility complex class II (MHCII)-associated invariant chain peptide (CLIP)-positive B cell
expansion after TBI. We also showed that antagonizing CLIP binding to the antigen presenting groove of MHCII after
TBI acutely reduced CLIP + splenic B cells and was neuroprotective. The current study investigated the chronic effects
of antagonizing CLIP in the 5xFAD Alzheimer’s mouse model, with and without TBI.
Methods 12-week-old male wild type (WT) and 5xFAD mice were administered either CLIP antagonist peptide (CAP)or vehicle, once at 30 min after either sham or a lateral fluid percussion injury (FPI). Analyses included flow cytometricanalysis of immune cells in dural meninges and spleen, histopathological analysis of the brain, magnetic resonancediffusion tensor imaging, cerebrovascular analysis, and assessment of motor and neurobehavioral function over theensuing 6 months.
Results 9-month-old 5xFAD mice had significantly more CLIP + B cells in the meninges compared to age-matched
WT mice. A one-time treatment with CAP significantly reduced this population in 5xFAD mice. Importantly, CAP also
improved some of the immune, histopathological, and neurobehavioral impairments in 5xFAD mice over the ensuing
six months. Although FPI did not further elevate meningeal CLIP + B cells, it did negate the ability of CAP to reduce*Correspondence:
M. Karen Newell-Rogers
mknewellrogers@tamu.edu
Lee A. Shapiro
lshapiro@tamu.edu
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
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Iannucci et al. Journal of Neuroinflammation(2024) 21:165

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meningeal CLIP + B cells in the 5xFAD mice. FPI at 3 months of age exacerbated some aspects of AD pathology in
5xFAD mice, including further reducing hippocampal neurogenesis, increasing plaque deposition in CA3, altering
microgliosis, and disrupting the cerebrovascular structure. CAP treatment after injury ameliorated some but not all ofthese FPI effects.
Keywords Alzheimer’s disease, Fluid percussion injury, Depression, Neurobehavior, Neuroinflammation,
Cerebrovascular, CD74, MHCII, Innate immune, Adaptive immune

Background
Traumatic brain injury (TBI) affects over 2–3 million
people annually in the United Sates and over 50 million worldwide [1, 2]. TBI is a significant risk factor for
Alzheimer’s disease (AD), the most common form ofdementia, increasing the risk for AD by 2.3–4.5-fold. Of
those who develop AD after TBI, the data suggest a lower
average age of disease onset [3, 4]. AD is characterized
by progressive memory loss and cognitive dysfunction,
which are also common features of TBI [5, 6]. Considering the increased risk for AD in the millions of people
who suffer a TBI each year, the 23 million Americans
over 40 with a reported history of TBI with loss of consciousness [7], and the estimated 6.5 million Americans
with AD in 2022 [8], it is vital to identify novel pathogenic mechanisms and therapeutic targets.
In both TBI and AD, innate immune and neuroimmune
activation have been reported in clinical and preclinicalstudies [9–13]. As part of the innate immune response,
glial cells and other immune cells are activated by, andrelease, pro-inflammatory cytokines and chemokines.
The TBI-induced inflammatory response can result ina transition to an antigen-specific adaptive immune
response [14–16], as evidenced by activation of immune
cells and antibody fragments of brain specific proteins [9,17]. An analogous adaptive immune response has also
been identified in both clinical and preclinical studies ofAD [18–20]. It is possible that adaptive immune components might link TBI and AD.
Accumulating evidence supports a pathogenic role foradaptive immune cell subsets in TBI and AD, including
B and T cell subsets [14, 21–24]. TBI pathology is augmented by activation of inflammatory T cells, including
expansion of CD8 + T cells and loss of regulatory T cells[16, 25]. In AD, T cells infiltrating into the CNS in bothclinical and preclinical models is also associated withworse outcomes [26–29]. The activation of T cells occurs
in response to antigen presentation by professional antigen presenting cells, including B cells.
TBI induces an increase in peripheral B cells after
injury [14, 30, 31], including altered B cell signatures in
the meninges [32]. Exogenous administration of purified naïve B cells is beneficial to TBI pathogenesis [33]. In
AD patients, reduced peripheral B cell subsets have been
detected [22, 23], and mature B cells have been identified in the AD brain [21, 34]. In a preclinical study using

three genetic AD mouse models, therapeutic depletion of
B cells was protective against AD-associated pathology[21]. Therefore, activation and expansion of B cell subsets
may represent a shared feature in TBI and AD that can betargeted to improve post-traumatic outcomes and alter
the course of AD pathogenesis. This notion is further
supported by the finding that depression and cognitive
impairment have been linked with altered B cell subsets[21, 35–37], and are common clinical syndromes associated with both TBI and AD [38–42].
Most TBIs, including preclinical models, are closed
head, sterile injuries. Thus, adaptive immune activation
after TBI may be an auto-immune response to TBI-generated self-antigens. Major Histocompatibility Complex
class II-associated invariant peptide (CLIP) occupies
the antigen binding groove of MHCII and plays a majormechanistic role in self-antigen presentation [43]. Previous studies have characterized a CLIP antagonist peptide(CAP) that preferentially targets CLIP + B cells [43–45].
Treatment with CAP after the fluid percussion injury(FPI) model of TBI was neuroprotective, anti-inflammatory, anti-neuroinflammatory, and acutely reduced
splenic CLIP + B cells [14]. Given the involvement of B
cells in both TBI and AD, as well as their role in activating
T cells, we hypothesized that antagonizing CLIP would
improve AD pathogenesis and ameliorate FPI-inducedchanges to the AD phenotype in the 5xFAD mice. To test
this hypothesis, a single dose of CAP was administered at
30 min after an FPI or sham injury in male 5xFAD mice.
Immune outcomes, neuroinflammation, AD-associatedneuropathology, and motor and chronic neurobehavioral
outcomes were assessed over the ensuing 6 months.

Results
Pathological assessments
A single dose of CAP selectively and chronically reduces
meningeal CLIP + B cells

Flow cytometric analysis of the meninges and underlying superior sagittal sinus and meningeal lymphatic(hereto referred to as meninges) [46] was performed. The
results showed that 9-month-old 5xFAD Sham + Vehicle(Veh) mice exhibited a significantly greater percent of B
cells (p < 0.001) and CLIP + B cells (P < 0.01), and a higher
expression level of CLIP on B cells (p < 0.05), comparedto age-matched WT Sham + Veh mice (Fig. 1B-D). In the
5xFAD mice, CAP given at 12 weeks of age had no effectIannucci et al. Journal of Neuroinflammation(2024) 21:165

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Fig. 1 CAP treatment results in a selective and chronic reduction in meningeal CLIP + B cells in 5xFAD mice, but not after FPI. In (A), the gating
strategy used to identify B and CLIP + B cells, T Cells, and macrophages. In (B), 5xFAD mice have significantly elevated B cells that are not changed by CAP
or by FPI. In (C) and (D), CLIP + B cells and CLIP expression on B cells are significantly elevated in 5xFAD mice compared to WT mice, and significantly reduced by CAP treatment given 6 months prior. The CAP treatment did not significantly reduce CLIP + B cells in the FPI mice (C, D). In (E), there was a significant decrease in CD3 + T cells in the meninges in 5xFAD mice that was not affected by CAP treatment or FPI. In (F), CD45high/CD19-/CD3- macrophages are
significantly elevated in 5xFAD mice, as is CLIP expression in these cells (G). CAP treatment did not have any significant effect on these CD45high/CD19-/
CD3- macrophages. Data are represented as Mean ± SEM; n = 3 for vehicle groups, n = 4 for CAP groups; *p < 0.05, **p < 0.01, **p < 0.001

Iannucci et al. Journal of Neuroinflammation(2024) 21:165

on the percent of meningeal B cells, but significantly
reduced the percent of CLIP + B cells (p < 0.05) and thecell surface expression of CLIP on B cells (p < 0.01), compared to age-matched 9-month-old 5xFAD Sham + Veh
mice (Fig. 1B-D). Conversely, in the 12-week-old 5xFAD
mice that received CAP at 30 min after FPI, CAP treatment failed to significantly reduce the elevated meningeal CLIP + B cells (Fig. 1B-D). Therefore, FPI negated theability of CAP to chronically reduce meningeal CLIP + B
cells in 5xFAD mice.
Further examination of meningeal immune cell types
showed that the 5xFAD Sham + Veh mice exhibited significantly reduced CD3 + T cells (p < 0.01; Fig. 1E), andsignificantly increased macrophages (p < 0.01), including CLIP + macrophages (p < 0.01), compared to WT
Sham + Veh mice (Fig. 1F-G). Neither CAP treatment
nor FPI had a significant effect on these immune subsets
in the 5xFAD mice (Fig. 1E-G). Thus, CAP specificallydepleted meningeal CLIP + B Cells in 5xFAD + Veh mice,but not 5xFAD FPI mice.
Peripheral immune cells from the spleens were also
examined from these mice. The results show that thepercent of CLIP + B Cells was significantly increased in
5xFAD Sham + Veh mice compared to WT Sham + Veh
mice (p < 0.01) (Fig. 2B). Unlike the meninges, there wasno chronic effect of CAP treatment on the percentageof splenic CLIP + B cells (Fig. 2B). Analysis of MHCII+/
CD11c + dendritic cells (DCs), CLIP + DCs, CD11c+/
Cd11b + macrophages, and CLIP + macrophages in the
spleen revealed no significant differences (Fig. 2D-G).
T cell analysis showed no significant differences in
the percentage of CD4 + T cells, although there wasa trend towards an increased percentage of CD8 + T
cells (p = 0.078) in 5xFAD Sham + Veh compared to WT
Sham + Veh (Fig. 2H-I). Compared to WT Sham + Veh
mice, 5xFAD Sham + Veh mice had a significantly
decreased percentage of CD8+/Ly49 + T cells (p < 0.05),
a population of CD8 + T cells recently described as regulators of autoimmunity [47, 48] (Fig. 2J). CAP treatment significantly reduced CD4 + T cells in the 5xFAD
mice (p < 0.05). No such effect was observed for CD8 + T
cells or CD8+/Ly49 + T cells in either CAP or FPI mice.
A notable finding was that both CAP treatment and FPI
increased CD44+/CD62L + central memory CD4 + T
cells, (p = 0.091, p = 0.079, respectively), and in 5xFAD
FPI + CAP mice, the increase in these central memory
CD4+ T cells was significant (p < 0.01). CAP treatment
and FPI significantly increased CD44-/CD62L + naïve
CD4 + T cells, compared to 5xFAD Sham + Veh mice(p < 0.05, p < 0.05, respectively) (Fig. 2K-O).

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Increased CD74 + cells in the hippocampus of 5xFAD mice isreduced by CAP

CD74 is a key immune regulator that is involved in boththe innate and adaptive immune response and is often
expressed at increased levels on multiple cell types underconditions of inflammation [49, 50]. CLIP is a proteolytic cleavage product of CD74 [49–51] that is produced in the lysosome [49]. CD74 has been implicatedin AD, and CD74 as well as its cell-surface ligand, MIF,
have been identified as potential therapeutic targets for
AD and TBI [14, 30, 52–56]. There are scant reports of
CD74 + cells in the brain, although in WT rodents theCD74 + cells appear to be largely restricted to the ependymal zones and choroid plexus [57]. Consistent withthese data, our analysis of CD74 + cells in the brains of
the WT Sham + Veh mice confirmed that the CD74 + cells
were largely restricted to the ependymal and choroid
plexus regions (not shown). Conversely, in the 5xFAD
mice, CD74 + cells were prominently observed in theependymal, choroidal, and parenchymal regions, including the neocortex and archicortex. Double-labeling of
the CD74 + cells in the hippocampus showed that noneof the CD74 + cells were GFAP+ (not shown), whereas
most were either macrophages or microglia, based on
double-labeling for anti-Iba1(Fig. 3A-B). The CD74 labeling appeared to be primarily around the perikaryal cytoplasm and the proximal processes (Fig. 3C).
Semi-quantitative analysis of the CD74 + cells in the
hippocampus demonstrated that they were significantly
increased in CA1 (p < 0.01) and the DG (p < 0.01) of the
5xFAD Sham + Veh mice compared to WT Sham + Veh.
There was also a trend towards an increase in area
CA3 (p = 0.055), indicating a robust genotype effecton the presence of hippocampal CD74 + cells (Fig. 3DF). CAP treatment significantly reduced the number ofCD74 + cells in 5xFAD sham mice in the DG (p < 0.05)
and trends in the reduction of these cells was observedin CA1 (p = 0.051) and CA3 (p = 0.061) (Fig. 3D-F). The
ability of CAP to reduce CD74 + cells in the hippocampus of 5xFAD mice is consistent with our previous study
showing that CAP reduces CD74 + splenocytes [14]. The
5xFAD FPI + Veh mice exhibited a significant reduction
in CD74 + cells in the DG (p < 0.05) and a trend in CA1(p = 0.051) compared to 5xFAD Sham + Veh mice. The
5xFAD FPI + CAP mice had significantly less CD74 + cells
in CA1 (p < 0.05) and the DG (p < 0.05).
CAP mitigates FPI-induced alterations in hippocampal CA3
plaque deposition in 5xFAD mice

Amyloid plaques are a pathological hallmark of AD
pathology and serve as a marker of AD-associated disease progression [58, 59]. The hippocampus is among
the first brain regions to exhibit plaques in clinicalAD [60] and the preclinical 5xFAD mouse model [61],Iannucci et al. Journal of Neuroinflammation(2024) 21:165

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Fig. 2 Effects of CAP and FPI on splenocytes in 5xFAD mice. In (A), there are no significant differences in splenic B cells. In (B), CLIP + B cells are significantly elevated in the spleen of 5xFAD mice compared to WT, but this elevation is not affected by CAP treatment or FPI. In (C), the gating for identifying
dendritic cells and macrophages. In (D) and (E), no significant differences were observed for dendritic cells (DCs) or CLIP + DCs. In (E) and (F), although
there were no significant differences in splenic macrophages, there was a trend towards an elevation of CLIP + macrophages in the 5xFAD mice thatwas not affected by CAP or FPI. Examination of CD4+ (H) and CD8+ (I) T cells revealed that CAP treatment significantly reduced CD4 + T cells in 5xFAD
mice, whereas the CD8 + T cells exhibited a trend towards an increase in the 5xFAD mice that was not influenced by CAP. Conversely, Ly49 + CD8 cells are
significantly decreased in the 5xFAD mice (J). Analysis of memory cell subsets (K-O) revealed that central memory CD4+ (M) and naïve CD4 + cells (N)were reduced in 5xFAD mice. CAP treatment, FPI, and FPI + CAP treatment all increased these populations of cells, suggesting that CAP and FPI could berestoring the ability of T cells to activate in the condition of chronic inflammation induced by the genotypic effects of the 5xFAD mice. Data represented
as Mean ± SEM; n = 3–4 per group; *p < 0.05, **p < 0.01

the latter of which begin to accumulate hippocampal
plaques as early as 2 months of age [62]. In the current
study, quantitative analysis of amyloid plaques in the
hippocampus confirmed previous reports showing subfield differences in plaque deposition in 5xFAD mice,
including differences in plaque size and number (Supp.
Figure 3C). When compared to age-matched 5xFAD
Sham + Veh mice, FPI increased the number of plaquesin CA3 (p < 0.05) (Fig. 4O), while CAP treatment after

FPI significantly reduced the number of plaques in CA3(p < 0.05) (Fig. 4O). Other hippocampal subfields, including the dentate gyrus and CA1, did not have FPI-inducedchanges in total plaque number (Fig. 4M-N) but did differ in size and distribution (Supp Fig. 3).
FPI alters microglia in the hippocampus of 5xFAD miceMicroglia are key mediators of neuroinflammation inboth TBI [63–65] and AD [11, 66, 67], and microglia canIannucci et al. Journal of Neuroinflammation(2024) 21:165

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Fig. 3 Significantly elevated CD74 + cells in the hippocampus of 5xFAD mice are reduced by CAP treatment and FPI. In (A-C), confocal micrographs of CD74 and CD74/Iba1 + cells in the hippocampus. The majority of CD74+ cells co-labeled with Iba1 (purple arrowheads), while a minority ofcells were not co-labeled (white arrowheads), indicating that most, but not all, CD74+ cells are likely macrophage/microglia. Analysis of CD74 cells in
the hippocampus revealed significant elevations in the DG (D), CA1 (E) and a trend towards elevation in CA3 (F) in 5xFAD mice compared to WT mice.
CAP treatment significantly decreased these cells in the dentate gyrus (D) and CA1 (E) and exhibited a trend towards a reduction in CA3 (F). Scale bars in
A-C = 30 μm, and 10 μm in D. Data are represented as Mean ± SEM; n = 3 for all groups; *p < 0.05, **p < 0.01

interact with peripheral immune cells, including B andT cells [68–71]. Consistent with previous data in 5xFAD
mice, we observed widespread increases in microglial number throughout the hippocampus compared
to WT mice (Fig. 5). There were significant increases
in the number of Iba1 + cells found in the DG (p < 0.05),
CA3 (p < 0.05), and a trend in CA1 (p = 0.069) (Fig. 5JL). While there were no effects of CAP or FPI in the DG
or CA1, in CA3 there was both a CAP and an FPI effect(Fig. 5L) whereby FPI significantly reduced the number ofIba1 + cells in CA3 (p < 0.01), and this decrease was significantly increased by CAP treatment (p < 0.05 vs. 5xFAD
FPI + Veh) (Fig. 5L). Thus, FPI reduced microglial cells
in CA3 and increased plaques in CA3, suggesting thatFPI 6 months prior may have altered the ability of CA3
microglial cells to phagocytose plaques. In support of thisnotion, CAP treatment after FPI increased the microglial
cells in CA3 and reduced the plaques. That CAP treatment significantly reduced CD74 + cells in the hippocampus, and CLIP + B cells, but not CLIP + macrophages in

the meningeal lymphatic, suggests a relationship between
CD74/CLIP, microglial cells, and plaque deposition.
Reduced adult hippocampal neurogenesis in the 5xFAD
mouse is improved by CAP and exacerbated by FPI

Reduced adult hippocampal neurogenesis has beenpreviously reported in 5xFAD mice at 2 [72], 4 [73, 74],
and 8 [75] months of age. Consistent with these data,
quantitative analysis revealed significantly less DCXlabeled immature granule cells in the DG of the 5xFAD
Sham + Veh mice (p < 0.05) compared to age-matched
WT Sham + Veh mice (Fig. 6F). CAP ameliorated the deficit in neurogenesis in 5xFAD sham mice but was unable
to rescue the reduction in 5xFAD mice that received an
FPI (Fig. 6F). These data mirror the effects of CAP andFPI on the meningeal CLIP + B cells, as seen in Fig. 1.
CAP improves chronic FPI-induced cerebrovascularalterations in 5xFAD mice

The cerebrovasculature has been found to be altered inclinical and preclinical AD [76–79]. Previous studies

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Fig. 4 Hippocampal β amyloid plaques in 5xFAD mice are altered by FPI and CAP in CA3. Representative micrographs of the DG (A-D), CA1 (E-H),
and CA3 (I-L) are shown. There were no significant differences in the DG (M), or in CA1 (N). Conversely, in CA3 there was a significant increase in 5xFAD
mice exposed to FPI (O), and CAP significantly prevents this FPI-induced increase in plaques. Scale bar in I = 100 μm for all micrographs. Data are represented as Mean ± SEM; n = 3 for vehicle groups, n = 6 for CAP groups; *p < 0.05

have identified chronic vascular alterations in 5xFAD
mice compared to age-matched WT, including reduced
vessel length and junction density [80]. The results fromthe current study show significantly altered cerebrovasculature after FPI in 5xFAD mice (Fig. 7). In the ipsilateral hemisphere after FPI in 5xFAD mice, there weresignificant reductions in junction density (p < 0.05), vessel density (p < 0.01), and average vessel length (p < 0.05),
as compared to 5xFAD Sham + Veh (Fig. 7D-F). CAP
treatment after FPI significantly increased vessel density(p < 0.05) and average vessel length (p < 0.05) compared tovehicle treated mice (Fig. 7D-F). No significant changes
were noted in the contralateral hemisphere (Fig. 7G-I).
Together, these data indicate that FPI chronically exacerbated cerebrovascular alterations in the 5xFAD mouse,
and that CAP treatment alleviated these effects.
CAP treatment restores altered brain connectome after FPI in
5xFAD mice

To further assess brain alterations in 5xFAD mice following FPI, we investigated cortical connectivity using
MRI (Fig. 8). There were clear genotype-related effects oncortical connectivity, such that sham 5xFAD mice exhibited reduced interhemispheric connectivity of the anterior cingulate cortex (ACC), compared to WT sham mice
(Fig. 8A). There was also an increased ipsilateral strengthof anterior to posterior connections between the ACC
and retrosplenial cortex (RSC) (Fig. 8A) in the 5xFAD
Sham + Veh mice. This latter effect was also observed in
5xFAD FPI + Veh mice (Fig. 8A). FPI + CAP treated mice
exhibited greater interhemispheric connectivity to the
RSC than the ACC (Fig. 8A), suggesting that CAP treatment enhances connectivity but not uniformly.
To better understand tracts entering the ACC, we generated tractography reconstructions by seeding the FPI
site above the dorsal hippocampus (Fig. 8B). Tract shapeanalysis revealed that 5xFAD mice had greater curl (total
length/Euclidean distance) in their tracts than their WT
counterparts (p < 0.05, Fig. 8B). FPI in 5xFAD Veh-treatedmice resulted in a trending increase in mean diffusivity(MD) (p = 0.07, Fig. 8B), with a concomitant increase inradial diffusivity (RD) (p = 0.05, Fig. 8B). Treating 5xFAD
mice with CAP after FPI significantly increased the number of streamlines relative to WT Sham + Veh controls

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Fig. 5 Increased hippocampal microglial cells in 5xFAD mice are reduced by FPI and restored by CAP in CA3. In (A-C), representative micrographsof Iba1 + microglia in CA3. In D-I, representative confocal z stacks depict co-staining for Iba1 + microglia and Aβ in 5xFAD FPI + Veh (D-F) and 5xFAD
FPI + CAP (G-I). Note, some of the plaques appear to have been phagocytosed by the microglial cells, as evidenced by plaques within the microglial
cytoplasm (purple arrowheads). Quantification of Iba1 + cells found a significant increase of microglial cells in the DG (J) and a trend in CA1 (K) of 5xFAD
mice that is not impacted by CAP or FPI. In L, the significant elevation of microglial cells in CA3 is reduced by FPI and restored by CAP treatment after FPI.
Scale bar in C = 50 μm for A-C, scale bars in D-I = 20 μm. Data are represented as Mean ± SEM; n = 3 for WT Sham + Veh, n = 6 for all 5xFAD groups; *p < 0.05,**p < 0.01

(p < 0.01, Fig. 8B). Overall, there was altered connectivity
from the FPI cortical site to the ACC in all FPI groups,
as compared to WT Sham + Veh. The 5xFAD Sham + Veh
and the 5xFAD FPI + CAP groups exhibited changes inshape analysis metrics [81], while 5xFAD FPI + Veh mice

had altered diffusivity metrics. These findings suggestthat ACC connectivity in both the 5xFAD Sham + Veh
and 5xFAD FPI + CAP groups was altered along the tracts(modified connectivity), while the 5xFAD FPI + Veh mice
had microstructural perturbations.

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Fig. 6 Reduced hippocampal neurogenesis in 5xFAD mice is improved by CAP and exacerbated by FPI. In (A-E), confocal micrographs illustrating DCX + immature neurons in the dentate gyrus of WT Sham + Veh (A), 5xFAD Sham + Veh (B), 5xFAD Sham + CAP (C), 5xFAD FPI + Veh (D), and 5xFAD
FPI + CAP (E). In F, quantitative analysis shows a significant reduction in DCX + cells in the 5xFAD mice that is rescued by CAP. These results also show thatFPI exacerbates the reduced hippocampal neurogenesis in 5xFAD mice, and CAP is unable to rescue this exacerbation. Qualitative analysis also reveals
alterations in dendrites and their branching through the granule cell layer into the molecular layer in 5xFAD mice (yellow arrowhead) compared to WT(white arrowheads), and this change is partially restored with CAP treatment (green arrowheads). These areas of the images are enlarged in the insets.
Data in F are represented as Mean ± SEM; n = 3–6; *p < 0.05, **p < 0.01

Fig. 7 FPI induces chronic alterations in cerebrovascular structure that are improved by CAP treatment. In (A-C), confocal micrographs of vessel
painting in 5xFAD Sham + Veh (A), 5xFAD FPI + Veh (B), and 5xFAD FPI + CAP (C). Quantification showed that FPI significantly reduced junction density, vessel density, and average vessel length in the ipsilateral hemisphere (D-F). Vessel density and average vessel length were significantly increased in FPI + CAP
mice (E, F). There were no significant changes identified in the contralateral hemisphere (G-I). Scale bars = 1000 μm. Data are represented as Mean ± SEM;n = 3 for all groups; *p < 0.05, **p < 0.01

Iannucci et al. Journal of Neuroinflammation(2024) 21:165

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Fig. 8 Altered connectivity in the 5xFAD mouse is affected by FPI and CAP treatment. In (A), connectogram analysis revealed genotype differences
between WT Sham + Veh and 5xFAD Sham + Veh mice, including reduced interhemispheric connections between the ACC (*). FPI in 5xFAD + Veh mice
showed exacerbated connectivity. In 5xFAD FPI + CAP mice there was a further reduction in bilateral ACC connectivity but increased (presumably compensatory) connections bilaterally in RSC. In (B), tract shape analysis revealed that 5xFAD mice have greater tract curl than their WT counterparts. In 5xFAD
FPI mice, CAP treatment significantly increased tract density relative to 5xFAD FPI + Veh- mice. In (C), 2D reconstructions of tracts illustrate the increased
number of tracts in 5xFAD FPI + CAP mice compared to the paucity of tracts in the 5xFAD FPI + Veh mice. Note the increased tract plasticity in the periinjury area. ACC – anterior cingulate cortex, BLA – basolateral amygdala, dHPC – dorsal hippocampus, IL – Infralimbic prefrontal cortex, PL – prelimbic
prefrontal cortex, RSC – retrosplenial cortex, vHPC – ventral hippocampus

Iannucci et al. Journal of Neuroinflammation(2024) 21:165

Additional assessment of tract alterations after FPI
and CAP treatment were visualized from 2D reconstructions of coronal sections at the injury site (Fig. 8C).
These images illustrate the dynamic reorganization of
the tracts (shown in orange) after injury on the ipsilateral hemisphere. In this case, this reorganization is more
pronounced in the 5xFAD FPI + CAP treated mice thanin the 5xFAD FPI + Veh mice. These findings suggestthat CAP treatment after injury in 5xFAD mice partiallyrestores the 5xFAD phenotype.
Behavioral assessments
CAP treatment improves acute gait deficits after FPI in 5xFAD
mice

Digigait testing was used to assess acute motor deficits.
Consistent with previous observations using this modelof FPI [82], 5xFAD FPI + Veh mice exhibited significantly
increased stance/swing on day 1 after injury (p < 0.05)
compared to 5xFAD Sham + Veh (Fig. 9A). This effectwas mitigated in the 5xFAD FPI + CAP group (Fig. 9A).
5xFAD FPI + Veh also showed a trending increase inabsolute paw angle (p = 0.076), showing that the righthind paw was turned outwards (Fig. 9B). The observed
preservation of motor function by CAP treatment after
FPI is consistent with our previous report showing that
CAP treatment reduces the hindlimb-associated corticallesion size after FPI in WT mice [14].
5xFAD mice exhibit altered affect that is partially improvedby CAP treatment, and aggravated by FPI

More than half of individuals who experience a TBI willdevelop depression, and depression may be a prodromeof AD [39, 83–85]. We investigated social interaction

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and burrowing activity beginning at 11 weeks of age andrepeated monthly for up to 130 days after FPI and CAP
treatment (Fig. 10). 5xFAD Sham + Veh mice exhibited a chronic deficit in burrowing compared to WT
Sham + Veh mice (p < 0.01) (Fig. 10A; Supp. Figure 1A).
This deficit was not improved in 5xFAD Sham + CAP
mice. In the 5xFAD FPI + Veh mice, there was a trend
towards a reduction in burrowing (p = 0.068) compared
to 5xFAD Sham + Veh at 10 DPI (Fig. 10A).
Social avoidance was assessed longitudinally using
the social interaction test [86, 87]. The results revealed
a significant effect of both CAP treatment (p < 0.001)
and injury (p < 0.001) in 5xFAD mice over the longitudinal course of the social interaction assessments. 5xFAD
Sham + CAP had a significant increase in social activity
over time (p < 0.05; Fig. 10B, Supp. Figure 1B), whereas
CAP treatment after FPI decreased social interaction
in 5xFAD FPI mice (Fig. 10B, Supp. Figure 1B). Exploratory analysis was performed to investigate associations
between neurobehavioral testing and pathological endpoints (See Supp. Figure 2 for full exploratory analyses).
A significant negative correlation was identified between
meningeal CLIP + B cells and the social index (r=-0.5625;
p = 0.0363), indicating increased CLIP + B cells were associated with reduced social interaction (Fig. 10C).
Cognitive impairment in 5xFAD mice can be altered by both
CAP and FPI

At 8 months of age, 5 months after FPI, mice underwent
cognitive behavioral testing in the pattern separation
test (PST) and the Barnes Maze to assess hippocampal associated learning and memory. In the PST, 5xFAD
Sham + Veh mice exhibit a reduced tendency to visit theFig. 9 CAP treatment mitigates FPI-induced acute motor deficits in 5xFAD mice. Mouse gait was analyzed using the digigait 1 day post injury
to assess motor deficits. 5xFAD FPI + Veh mice had significantly increased stance/swing (A) and a trending increase in paw angle (B) in the right hindleg, indicating an overall change in motor function after injury. These deficits were not apparent in the 5xFAD FPI + CAP mice. Data are represented as
Mean ± SEM; n = 6 for vehicle groups, n = 12 for CAP groups; *p < 0.05

Iannucci et al. Journal of Neuroinflammation(2024) 21:165

Page 12 of 24

Fig. 10 Differential effects of CAP on affect-associated behavior. In (A), 5xFAD mice exhibit reduced burrowing behavior compared to WT mice, including a significant reduction at 70 DPI and a trend at 130 DPI. Burrowing was reduced after FPI in 5xFAD mice at 10 DPI, but this was not altered by CAP
treatment. In (B), CAP treatment differentially affects social interaction in 5xFAD mice. CAP increased social interaction in 5xFAD sham mice, most notably
at 70 DPI, but reduces social interaction in 5xFAD FPI mice such that 5xFAD FPI + CAP mice exhibited significantly reduced interaction compared to 5xFAD
Sham + CAP mice at 70, 100, and 130 DPI. In (C), regression analysis revealed a significant negative correlation between social interaction and meningeal
CLIP + B cells. Data are represented as Mean ± SEM; n = 6 for vehicle groups, n = 12 for CAP groups; *p < 0.05, **p < 0.01, ***p < 0.001

novel object (NO) when compared to WT Sham + Veh(66.67% vs. 100%, respectively), and WT Sham + Veh
mice exhibited a significant preference for the NO
over the FO (p < 0.05) (Fig. 11A-B). Neither the 5xFAD
Sham + Veh mice, nor the 5xFAD FPI + Veh mice showed
any NO preference (p = 0.132, p = 0.9326, respectively),indicating a failure to recognize the novel object. Further analysis revealed that CLIP + B cells in the duralmeninges had a significant negative correlation with thenumber of visits made to the novel object in the PST (r=0.409; p = 0.0395; Fig. 11C), indicating that CAP depletionof meningeal CLIP + B cells is associated with improved
PST performance in 5xFAD Sham mice, and suggestingthat CLIP + B cells may be a critical population in AD
pathogenesis and cognitive impairment.
Consistent with previous studies [88], 5xFAD
Sham + Veh mice exhibited impairment in the Barnes
maze test (Fig. 11D). This was notable during the first
four-days of training where there was a significant
increase in average escape latency compared to WT
Sham + Veh (p < 0.01) (Fig. 11D). This impairment was
not improved by CAP treatment or altered by FPI. There
were no significant effects identified in CAP or FPI miceon performance in probe trials. These findings suggest apossible floor effect in the 5xFAD mice at this advanced
stage of disease progression.

Discussion
In this study, the pathological and behavioral effectsof CLIP antagonism in 5xFAD mice were examined at
6-months after either sham or FPI. The first notablefinding is that in the spleen and meninges, CLIP + cells,
including CLIP + B cells, are elevated in 9 month-old
5xFAD mice, when compared to age-matched WT mice.
A one-time treatment with CAP at 3 months of age significantly depleted the meningeal CLIP + B cell population, but not macrophages, CLIP + macrophages, or
splenic CLIP + B cells. Interestingly, the extent of meningeal CLIP + B cells was correlated with decreased social
interactions and impaired performance in the PST.
FPI in 5xFAD mice did not chronically alter the B
cells, macrophages, dendritic cells, CD4 + or CD8 + T
cells in the spleen or meninges. However, it did increase
the naïve and central memory CD4 + T cells relative to
5xFAD Sham + Veh mice. These increases may reflect
TBI-driven activation and expansion of naïve CD4 + T
cells, or TBI-driven reactivation of memory CD4 + T
cells. A similar increase in naïve and central memory
CD4 + T cells was seen in CAP treated 5xFAD mice. In
parallel, CAP treatment seemed to improve some aspectsof behavioral performance, including social interaction.
It is important to note that the 5xFAD mice exhibit arobust behavioral impairment, particularly in cognitive
behavioral testing, and the lack of chronic exacerbation
by FPI may be a result of floor effects in some aspects

Iannucci et al. Journal of Neuroinflammation(2024) 21:165

Page 13 of 24

Fig. 11 5xFAD mice exhibit hippocampal-associated cognitive deficits. In (A-C), mice were challenged with the pattern recognition test (PRT). The
results showed that a reduced percentage of 5xFAD Sham + Veh mice visit the NO first (A), and this effect was exacerbated by FPI. In (B), WT mice showeda significant preference for the NO that was not found in any of the 5xFAD groups. In (C), visits to the NO in PRT exhibited a significant negative correlation
with the percentage of meningeal CLIP + B cells. In (D), 5xFAD mice had a longer average escape latency in the Barnes maze during the 4 training days,
but this is not changed by FPI or CAP treatment. No significant differences were identified in the probe trials (E-G). It is pertinent to note that several mice
did not visit the target quadrant for the full 3 min. Data are represented as Mean ± SEM; n = 6 for vehicle groups, n = 12 for CAP groups; *p < 0.05, **p < 0.01

of neurobehavioral performance. It is also possible that,as has been previously reported in FPI models, some
aspects of neurobehavioral impairment may be transient.
That CAP improved some neurobehavioral outcomes insham 5xFAD mice suggests that stimulating the transition from an innate to an adaptive inflammatory response
early in the pathological progression of 5xFAD mice may
ameliorate the adverse effects of chronic inflammationand modify the pathological progression.
While the precise mechanisms of the current findings
remain elusive, the observation of altered memory andnaïve CD4 + T cells is highly suggestive of the possibility
that both CAP and FPI promote antigen processing andpresentation by professional antigen presenting cells to
T cells, leading to an antigen-specific adaptive immune
response. This possibility is supported by the expansionof CLIP + B cells, as well as the trends towards increases
in naïve and central memory CD4 + T cells in CAP and
FPI mice. That the cumulative effect of CAP and FPI
resulted in a significant elevation of these T cell subsets

also supports this notion. In the case of CAP, which was
designed to outcompete CLIP for the MHCII antigen
binding groove [43], it is possible that CAP itself waspresented and recognized by T cells, as CAP has beenpreviously shown to promote the transition to an adaptive immune response [43, 44]. However, CAP has also
been shown to have anti-inflammatory properties following FPI and these can’t be ruled out as possible therapeutic mechanisms of the present study [14]. Still, CAP
inhibited the chronic expansion of some inflammatory
immune cell subsets, expanded central memory andnaïve T cells, and rescued some of the well-describedneuropathology and cognitive deficits in the 5xFAD
mice. Future studies are needed to more fully elucidate
the immune mechanisms by which CAP exerts beneficial
effects.
Another novel discovery in the current study is theidentification of CD74 + cells in the hippocampus of
5xFAD mice. The hippocampus is a major component incognition, learning, and memory, and is a primary area

Iannucci et al. Journal of Neuroinflammation(2024) 21:165

of pathology after TBI, in AD, and in the 5xFAD mousemodel [58, 62, 89–96]. CD74, of which CLIP is a cleavage product, has been shown to increase expression on
both immune and non-immune cells under conditions ofinflammation [50]. However, the role of CD74 + cells thathave entered the brain parenchyma has not been previously explored. Therefore, future studies are also neededto address the potential role of CD74 + cells in the hippocampus of 5xFAD mice.
The FPI model, like the vast majority of clinical TBIs,
is a closed head injury in which foreign antigens are notintroduced into the brain. Therefore, the antigen-specificadaptive immune response reported in preclinical andclinical TBI studies [14, 15, 97] is highly likely to be presenting self-antigens. This is supported by the previous
observations of brain specific antibodies in the CSF andserum of TBI patients [98, 17, 99–103]. In the current
study, one of the neuropathological changes induced by
FPI was the long-term exacerbation of decreased hippocampal neurogenesis observed in the 5xFAD mice. Adult
hippocampal neurogenesis has been linked to patternseparation performance [104] and the PST deficit wasalso exacerbated by FPI. Pattern separation deficits havebeen previously reported in human AD patients [105–107], and in mouse models of AD [108], including 5xFAD
mice. In the latter 5xFAD model, impaired hippocampal
neurogenesis has been reported as early as 2 months ofage [109]. It is interesting to consider that CA3 pyramidalcells are targets of the dentate granule cell mossy fibers.
The accumulation of plaques in CA3 observed in the current study may therefore alter the mossy fiber growthcues leading to reduced generation of newborn granulecells, and this may link the altered adult neurogenesis tothe impaired PST performance.
The decrease in microglial cells and increase in plaquedeposition after FPI in CA3 of 5xFAD mice is of particular interest and may further implicate CA3 in the hippocampal-related cognitive impairment. CA3 has been
linked to Barnes Maze performance [110–113], among
other components of spatial learning and memory. One
possible interpretation of these findings is that FPI in
the 5xFAD mice induced a loss of microglial cells in CA3
that enabled the plaques to increase in size and number.
This notion is supported by previous studies that indicate microglial cells can phagocytose plaques and thatmicroglia alter their expression profile in response toplaques [114–116]. Additionally, MHCII + microglia havebeen implicated in both AD and experimental TBI [117,118], and the presence of the CD74 + cells also cannot be
ruled out as contributing to the loss of Iba1 + cells. Previous studies using the FPI model have also identified
pathological alterations to CA3, and further examinationof the potential role of CA3 in post-traumatic syndromes
is needed.

Page 14 of 24

The cerebrovascular system is a critical nexus throughwhich immune cells interact with the brain [78], and vascular permeability or damage may facilitate the entry of
immune cells into the brain parenchyma. Indeed, cerebrovascular damage and dysfunction in both TBI and
AD have been linked to immune cell activation andinflammation, including increased migration of peripheral leukocytes into the brain [119, 120]. FPI in clinicaland preclinical studies results in acutely altered vascularmorphology [121, 122]. Vascular dysfunction is also present early in the pathogenesis of AD, and vascular alterations have been identified in the 5xFAD model [76, 80].
Here, CAP treatment attenuated the chronic FPI-related
cerebrovascular changes in 5xFAD mice, highlighting thepotential crosstalk between the cerebrovasculature andthe activated immune system in the progression of ADassociated pathology after injury.
The observation of alterations in cortical connectivity between 5xFAD mice and age-matched WT mice isconsistent with previous clinical and preclinical AD and
TBI studies showing altered connectivity between brain
regions [123–126]. Novel to our study was the clear genotype-related effect, with 5xFAD exhibiting reduced cortical interhemispheric connectivity, but increased strengthof anterior to posterior connections compared to WT.
FPI further altered cortical connectivity in 5xFAD mice,and this was modulated by CAP treatment. For example,
FPI associated decreases in connectivity between bilateral ACC regions was decreased in 5xFAD mice with
CAP, but compensatory increases to the bilateral RSCRSC connections were increased, as was ipsilateral ACCRSC connectivity. The ACC is involved in higher-level
functions, including reward anticipation, decision making, and emotion, and in cognitive tasks [127]. The ACC
connects broadly to multiple regions including the RSC
and is also engaged in higher-level cognitive tasks, inparticular spatial cognition [128]. That these circuits arealtered in 5xFAD mice, and can be further modulated by
FPI and CAP, may provide further insight into the deficits
identified in neurobehavioral tasks in 5xFAD mice bothwith and without injury and highlight the importance of
immune regulation in these processes.
Taken together, antagonism of CLIP was found to differentially impact AD-associated pathology in 5xFAD
mice with and without TBI. In 5xFAD mice without TBI,
CLIP antagonism reduced meningeal CLIP + B cells andhippocampal CD74 + cells, enhanced neurogenesis, andimproved some aspects of neurobehavioral dysfunction.
On the other hand, CLIP antagonism did not affect meningeal CLIP + B cells after FPI in 5xFAD mice or improve
most behavioral dysfunction. However, CLIP antagonism
after FPI did improve TBI-induced neuropathologicalchanges, including plaques, microglia, cerebrovascularalterations, and altered cortical connectivity. The lack of

Iannucci et al. Journal of Neuroinflammation(2024) 21:165

Page 15 of 24

significant exacerbation by FPI on behavioral impairment
may be due to the strong behavioral impairment exhibited in the 5xFAD mice, and/or the transient nature ofcognitive impairment after TBI and in preclinical models of TBI. Nevertheless, this study identified meningeal
CLIP + B cells as a possible pathogenic mediator in AD
pathogenesis, including after TBI. Future studies areneeded to more fully investigate the mechanisms responsible and the possible therapeutic implications of targeting this unique immune cell population.

animal use protocol #2020 − 0140. Male Wildtype (WT)
C57bl/6J (Jackson Laboratory, Stock #000664) and
5xFAD mice (MMRRC034848 B6.Cg-Tg (APPSwFlLon, PSEN1*M146L*L286V)6799 Vas/Mm) (male, age 9
weeks) were purchased from the Mutant Mouse Resource
and Research Center (MMRRC) at Jackson Laboratory
and allowed to acclimate for 1 week prior to the experimental start. All mice were housed individually in ventilated cages in a controlled environment and maintainedon a standard diet for the duration of the experiment.

Materials and methods

Fluid percussion injury (FPI)

Experimental design

Lateral FPI was used as a model of TBI and was performed as previously described [14, 30, 129, 130]. Briefly,
a 2 mm craniotomy, performed using a stereotaxic device(Stoelting, Wood Dale, IL) under anesthesia, was performed over the left parietal cortex, keeping dura intact.
The female end of a luer-lock syringe was cemented overthe craniotomy and attached to the FPI apparatus (Custom Fabrication & Design, Richmond, VA). A 12–16 ms
FPI was delivered at a pressure of ~ 1.5–1.8 atm. Shammice received identical treatment, with the exception
being that no pressure pulse was delivered. Mice weremaintained under isoflurane anesthesia for the durationof the surgical procedure, except during the impact.

The experimental design proceeded as depicted inFig. 12. Following one week of acclimation training, baseline depression testing was performed on all mice at 11
weeks of age. Subsequently, male 5xFAD mice received
either lateral FPI as a model of TBI or sham surgery at
12 weeks of age. Thirty minutes after injury, mice weretreated once with a single intraperitoneal (i.p.) dose
(1 mg/kg) of CAP or Veh. An additional group of agematched WT male mice received sham injury and vehicle
treatment, as a genotype control. For behavioral testing, groups were n = 6 for vehicle treatment groups andn = 12 for CAP treatment groups. All mice underwent
acute monitoring of motor deficits, followed by depression testing 10 days post injury (DPI), and then every 30
days for the next 120 days. At 150 DPI (32 weeks of age),cognitive performance was assessed using the patternseparation test (PST) and Barnes Maze. At 180 DPI (36weeks of age), the mice were euthanized, and tissue wascollected for analysis. A subset of mice (3–4 per group)
was used for flow cytometric analysis and others wereused for magnetic resonance diffusion tensor imaging(MRI DTI), vessel painting, and immunohistochemistry(3–6 per group).
Animals

All work was approved by the Texas A&M Institutefor Animal Care and Use Committee (IACUC) under

Drug Treatment

Mice were treated with CAP as previously described [14,30]. Mice were injected i.p. with 1 mg/kg CAP dissolved
in dimethyl sulfoxide (DMSO) and further diluted withsterile saline. Vehicle mice received equivalent volumeof DMSO dissolved in sterile saline. All treatments wereadministered 30 min after the FPI or sham procedure.
Digigait

Our FPI model consistently yields a transient contralateral hindlimb deficit that resolves within 5–7 days
after injury. To confirm this deficit, the digigait system (Mouse Specifics Inc., Framingham, MA) was used
to assess acute changes in gate for the first 5 days afterFig. 12 Experimental design. Male 12-week-old wild type (WT) or 5xFAD mice received either fluid percussion injury (FPI) or sham surgery followed
30 min later by treatment with the CLIP antagonist peptide (CAP) or vehicle. Gait analysis was done at 1–5 days post injury (DPI). Depression-associatedbehavioral testing was done at 10 DPI and every 30 days thereafter. Cognitive testing started at 150 DPI. Tissue collection was done at 180 DPI when themice were 9 months of age, and tissue was used for a number of pathological analyses including immunohistochemistry (IHC), flow cytometry, vessel
painting (VP), and diffusion tensor imaging (DTI)Iannucci et al. Journal of Neuroinflammation(2024) 21:165

FPI or sham procedure in 5xFAD mice. Mice were acclimated to the room and trained to walk on the digigait at
12 cm/s 3 days prior to surgery. Following FPI or sham,mice walked on the digigait every day for the first 5 DPI,
and videos were recorded for analysis. Analysis was doneusing the digigait software (Mouse Specifics Inc., Framingham, MA), and metrics of interest included paw angle
and stride/swing.
Depression-Associated behavioral testing

The presence of a depression-associated phenotype isoften described using multiple tests, since no single test
is considered declarative [131–134]. Mice underwent
testing for depression-associated behaviors including asocial interaction test and burrowing test, modified fromprevious studies in rats [135, 136]. All mice received 2days of acclimation for each test at 10 weeks of age, andthen one week later underwent baseline testing for all
tests. The same depression testing was then repeated at
10, 40, 70, 100, and 130 DPI for all mice.
Burrowing

The burrowing test has been used as a measure of fatigue
and anhedonia [137], common symptoms of humandepression identified by the Diagnostic and Statistical Manual of Mental Disorders (DSM) V [138], as well
as of spontaneous activity/voluntary behavior [135, 139,140], and has therefore been related to the animal’s overall well-being and a depression-like phenotype in rodents[141]. Burrowing tubes were created using white PVC
pipe (2” diameter), all tubes were 5” long. For testing,tubes are filled up to 4” with corncob bedding and thenplaced in the corner of the animal’s home cage for 15 min.
Mice are allowed free access to the burrowing tube for
15 min, after which the tubes were removed. Tubes are
Table 1 Antibodies for immunohistochemistry
Antibody

Species

Dilution

Iba1

Rabbit

1:400

Aβ
Aβ
CD74

Rabbit
Mouse
Rat

1:500
1:500
1:200

DCX (C)DCX (N)
Anti-rabbit
AlexaFluor-555
Anti-rabbit
AlexaFluor-488
Anti-Rat
AlexaFluor-555
Anti-Goat
AlexaFluor-555

Goat
Goat
Goat

1:200
1:200
1:200

Company (Catalog#)
Wako Labs(019-19471)
Cell Signaling (8243)
BioLegend (803,001)
BD Pharmingen(555,317)
Santa Cruz (sc-8066)Santa Cruz (sc-8067)
Invitrogen (A21428)

Goat

1:200

Invitrogen (A11070)

Goat

1:200

Invitrogen (A21434)

Donkey1:200

Invitrogen (A21432)

Page 16 of 24

weighed before and after testing, and the amount ofbedding burrowed out of the tube is calculated. Prior to
testing all animals were acclimated once to the empty
burrowing tube for 1 h and once to a full burrowing tube
for 15 min to ensure interest in the tube and task.1
Social interaction test

Social withdrawal is a common element of humandepression [142, 143]. In rodents, the social interaction
test can be used to assess social avoidance [86, 144].
The social interaction test was performed as previously
described with some modifications [87, 145–147]. The
test mice were each acclimated to the testing box, a white
plexiglass open field box (60 cm x 45 cm), for 5 min.
Following acclimation, a social mouse (age matched
C57Bl6/J) was added to the testing apparatus within acircular wire cage placed against the wall in the middleof the long side of the open field box. The test mouse wasallowed to freely explore the test apparatus for 5 min, andthe number of interactions made with the social mouseand time spent interacting with the social mouse wererecorded. The corners opposite to the social mouse cagewere designated as avoidance zones, and time spent inavoidance and number of visits to avoidance zones werealso manually quantified. A social index was calculated
as the amount of time spent in social zones minus timespent in avoidance zones divided by the total time spentin social and avoidance zones.
Neurobehavioral testing

At 32 weeks of age, all mice underwent neurobehavioral
testing to assess possible cognitive impairment in tasks
related to the hippocampus, the pattern separation test
(PST) and Barnes Maze.
Pattern separation test (PST)The PST was used to measure the ability of mice to recognize pattern separation, as previously described [148–150]. The PST was incorporated into this study to assessa hippocampal-dependent task that is linked to intact
neurogenesis [104]. The test was comprised of 3 successive trials separated by a 1-hour intertrial interval. Mice
were previously acclimated to the open field test box,
as described above for the social interaction test. In thefirst trial, the animal was placed in the open field box
with a first set of two identical objects (shape 1 objects)
positioned on floor pattern 1 (P1) and was allowed tofreely explore both objects for 5 min. In the second trial,
mice were placed in the open field box with a second setof identical objects (shape 2 objects) on floor pattern 2(P2) and again allowed to freely explore both objects
for 5 min. In the third and final trial, one of the shape 2
objects from trial 2 was replaced with a shape 1 object
on P2. This shape became the novel object (NO) andIannucci et al. Journal of Neuroinflammation(2024) 21:165

shape 2 object became the familiar object (FO) on theP2 floor. Again, the mice were allowed to freely explore
both objects for 5 min. Each trial was video recorded
and analyzed using automated NOLDUS EthoVisionXTvideo tracking software. Additional scoring was done
manually, in which the visits counted to each object werecounted. Along with the number of visits to each object,
the latency to visit the NO and the first object visited byeach mouse was recorded.
Barnes Maze

The Barnes maze is used as an assessment of spatial
learning and memory in rodents [88, 151, 152]. Briefly,
the maze consists of a brightly lit, white elevated circular
platform with equally spaced holes along the perimeter.
A non-visible escape box is placed under one of the holes.
Mice completed a habituation trial to become familiar
with the maze and the environment, and practice entering into the escape box. In the learning phase, mice weretrained to find the escape hole. Mice completed 2 trials
each day, separated by a 15 min intertrial interval, for 4
consecutive days. Each trial was a maximum of 3 min.
If successful, the mouse was rewarded with 1 min in the
covered escape box in the dark. If not successful, themouse was guided to the escape box at the end of 3 min
and allowed to sit for 1 min in the dark. The number ofincorrect holes visited (errors) as well as the latency tofind the escape box was recorded for each trial and averaged for each day for each mouse. 72 h after the lasttraining trial, a probe trial in which the escape box was
removed was conducted. During the probe trial, mice
were allowed to freely explore the maze for 3 min. Eachtrial was video recorded and analyzed using automated
NOLDUS EthoVisionXT video tracking software. Additional scoring was done manually, in which latency to theescape hole and visits to additional holes were counted.
Immunohistochemistry

Immunohistochemistry for doublecortin (DCX) + immature neurons, Iba1 + microglia, CD74 + cells, andamyloid-β plaques was used, as previously described[148, 153–155]. Briefly, mice were anesthetized with
Fatal Plus (Sodium Pentobarbital; 52 mg/kg, administered i.p.) and transcardially perfused with phosphatebuffered saline (PBS) through the left ventricle until the
blood ran clear. This was followed by 4% paraformaldehyde (PFA) through the left ventricle. All brains wereallowed to postfix in the skull for 24 h in PFA, after which
they were extracted and fixed for an additional 24 h in
4% PFA. Fixed brains were cut into 44-µm thick serial
sections with a freezing microtome (American Optical
Corp; Model #860). For DCX and CD74, slices were first
incubated in a 1X Citrate Buffer (Millipore Sigma) for 1 h
at 45 °C. Slices were then washed and stained with goat

Page 17 of 24

anti-DCX antibody (Santa Cruz Inc. USA), rat anti-CD74(BD Biosciences), rabbit anti-Iba1antibody (Fujifilm
Wako Chemicals USA Corp), or rabbit anti-amyloid-β(Cell Signaling Technology) overnight at room temperature, rotating. After overnight incubation, slices were
washed and stained with and secondary biotinylated goatanti-Rabbit IgG (Alexaflour-555) (Iba1, amyloid-β), secondary donkey anti-Goat IgG (AlexFlour-555) (DCX), or
secondary biotinylated goat anti-Rat IgG (Alexfluor-555)(CD74). Antibodies used are shown in Table 1 Slices
were mounted and cover-slipped with antifade reagent
containing DAPI (Vector Laboratories #H-1200-10). For
reference, we stained 3-month-old WT mice (Fig. 13)
simultaneous with all other tissue to confirm the efficacyof our previously described DCX-staining protocol [94,148, 153, 155].
Quantitative and semiquantitative analysis

Imaging for all immunohistochemistry was done on
a fluorescent microscope (Olympus, Bethlehem, PA).
Unbiased stereology-based analysis was used to quantify
cells positive for DCX, Iba1, or amyloid-β plaques in the
hippocampus, as previously described [30, 94, 148, 153,
156]. Sections (~ every 260–350 μm apart) containing
the dorsal hippocampus (Bregma − 1.34 through − 2.80)
were selected for analysis. Analysis of DCX + cells was
performed in the ipsilateral infra- and supra-pyramidal
blades of the DG granule cell layer/subgranular zone, aspreviously described [94, 148]. Iba1 and amyloid-β analyses were done in CA1, CA3, and the dentate gyrus (DG)
hippocampal subregions in the ipsilateral hemisphere.
A minimum of 3 left hippocampi were counted per animal, per antibody, within the stereological coordinates
indicated above. We also performed semi-quantitative
assessment of CD74 + cells in the ipsilateral hippocampus. Slices were scored on a scale of 1–4, and a minimumof 3 slices per animal were evaluated. We further sought
to qualify the identification of the CD74 + cells by double-staining with either GFAP for astrocytes or Iba1 formacrophage/microglia.
Flow cytometry
Dural meninges collection and isolation

Mice were perfused with PBS for approximately 5 min,until liver was cleared and the blood ran clear. The skull
was rapidly removed and meninges (dura mater, arachnoid, and pia mater) along the superior sagittal sinus
were collected using forceps and placed in PBS + 10%
Fetal Bovine Serum (FBS) on ice while remaining samples were collected. Following collection, solutions ofdigestion enzymes containing collagenase 4 and DNAse
1 were prepared. All dura were washed once with PBS,
then incubated in digestion enzymes for 1 h at 37 °C.
At the conclusion of 1 h, the reaction was quenched by

Iannucci et al. Journal of Neuroinflammation(2024) 21:165

Page 18 of 24

Fig. 13 DCX staining in 3 month old WT mouse. To confirm the efficacy of our DCX staining protocol, we stained tissue from a 3 month old WT control
mouse and found normal, robust DCX staining in the GCL of the dentate gyrus. This confirms our protocol and highlights the low number of DCX+ cells
in our aged miceadding the enzyme mix to PBS + 10% FBS. All samples
were subsequently spun and resuspended in the desired
buffer for flow cytometry.
Spleen collection and isolation

Splenocytes were isolated as previously described withminor modifications [14, 30]. Briefly, mice were euthanized, and spleens were removed. Tissues were dissociated, single cell suspensions prepared, and red blood
cells were lysed using ACK lysis buffer. The dissociatedsplenocytes were then counted and stored for subsequent
analysis via flow cytometry.
Staining of single cell suspensions

Splenic and meningeal cells were analyzed by flow
cytometry as previously described [14, 30]. All cells weretreated with FC Block (BD Bioscience) prior to staining.
Meninges were stained with the following antibodies:
CD19 (6D5, BioLegend), CD90.2 (53 − 2.1, BioLegend),
CD45 (30-F11, BioLegend), and CLIP (15G4, Santa Cruz
Biotechnology). Due to the limited number of cells foundin the meninges, only limited immune cell subsets could
be examined. More expansive immune cell analysis was
performed on splenocytes from these same mice. These
included antibodies to the following immune cell subsets:
CD19 (6D5, BioLegend), CD90.2 (53 − 2.1, BioLegend),
CLIP (15G4, Santa Cruz Biotechnology), CD4 (RM4-5,
BioLegend), CD8 (53 − 6.7, BioLegend), CD44 (IM7, BioLegend), CD62L (MEL-14, BioLegend), Ly49 (RA3-6B2,

BioLegend), Cd11b (M1/70, BioLegend), Cd11c (N418,
BioLegend), and MHCII (M5/114.15.2, BioLegend). Livecells were assessed using Ghost Dye Red 780 viability dye(Tonbo Biosciences). The cells were analyzed using a Becton Dickson Fortessa X-20 flow cytometer and data wereanalyzed using FlowJo software (TreeStar Inc.) Antibody
details for flow cytometry are found in Table 2.
Vessel painting methods and analysis

Analysis of the cerebrovasculature was done using vessel
painting methods and analysis as previously describedwith slight modification [79, 80, 157]. Mice were injected
i.p. with heparin and sodium nitroprusside (SNP) andwere anesthetized with an i.p. injection of Fatal Plus
(Sodium Pentobarbital; 52 mg/kg). Vessel painting was
performed by injecting a solution of 1,1′-dioctadecyl3,3,3′3′-tetramethylindocarbocyanine perchlorate (DiI)(0.3 mg/mL in PBS containing 4% dextrose, total volumeof 500 µL) into the left ventricle, followed by a 50 mL PBS
flush and a 50 mL 4% PFA perfusion, using a peristaltic
pump (8.4 mL/min). The brains were post-fixed in theskull for 24 h, then the brains were extracted, and fixed
for an additional 24 h in 4% PFA, washed in PBS, andstored at 4 °C in PBS until imaging. Successfully vessel
painted brains were selected if they showed uniform pinkstaining and excellent staining of large and small vessels
on the cortical surface, as previously described [157].
The brains were imaged using a fluorescence microscope (Keyence BZ-X810, Keyence Corp, Osaka, Japan).

Iannucci et al. Journal of Neuroinflammation(2024) 21:165

Table 2 Antibodies for flow cytometry
Antibody

Clone

GhostDye(Live/Dead)

Fluorophore

Dilution

Red780

1:100

CD19

6D5

PE

1:200

CD19

6D5

BV421

1:200

CD3

145-2C11

BV421

1:200

CD90.253-2.1

BV510

1:200

CLIP

15G4

FITC

1:100

MHCII

M5/114.15.2 PerCp-Cy5.5

1:100

CD11c

N418

PE

1:100

CD11b

M1/70

BV711

1:200

CD45

30-F11

BV605

1:300

CD4

RM4-5

BV605

1:100

CD8α

53-6.7

BV711

1:100

CD44

IM7

PE

1:20

CD62L

MEL-14

BV421

1:100

Ly49
14B11
C/F/I/H
CD45R/
RA3-6B2
B220
CD16/CD32 2.4G2(FC Block)

PE/Cy7

1:100

BV785

1:300

N/A

1:50

Company(Catalog #)Tonbo
Biosciences(13-0865)
BioLegend(115,507)
BioLegend(115,538)
BioLegend(100,335)
BioLegend(140,319)
Santa Cruz
(sc-53,946)
BioLegend(107,624)
BioLegend(117,308)
BioLegend(101,241)
BioLegend(103,140)
BioLegend(100,547)
BioLegend(100,747)
BioLegend(103,024)
BioLegend(104,435)
BioLegend(108,209)
BioLegend(103,246)
BD Pharmingen (553,142)

Axial images of the entire brain were acquired at 2× magnification using the Z-stack feature (~ 42 images, step
size 25.2 μm). Classical vessel analysis was performed byusing the Angiotool software (Version 0.6a), allowing formeasures of vessel density, length, and number of junctions [158], and the ImageJ plugin “FracLac” was used
to analyze vascular complexity through fractal analysis[159].
Magnetic resonance imaging (MRI) via diffusion tensor
imaging (DTI)
MRI acquisition

Four groups of mouse brains (WT Sham + Veh, 5xFAD
Sham + Veh, 5xFAD FPI + Veh, and 5xFAD FPI + CAP; 6
per group) were imaged on a 9.4T Bruker Avance imager(Paravision 5.1, Bruker Biospin, Billercia, MA). Diffusion
tensor imaging (DTI) and T2-weighted imaging (T2WI)
scans were acquired for 1 h 52 min and 38 min, respectively. Both scans were acquired with the same spatial

Page 19 of 24

resolution: 1.5 cm field of view, 0.5 mm slice thickness,
and a 128 × 128 acquisition matrix. DTI images were collected with repetition time/echo time (TR/TE) = 8000
msec/35.7 msec, and 30 directions (5 b = 0mT/m,
b = 3000mT/m). T2WI scans were acquired with 10
echoes and TR/TE = 4000 msec/10 msec.
MRI analysis

Experimenters were blinded to group designations during MRI analysis. The skull was stripped from the scanusing ITK-SNAP (version 3.6.0) [160]. DTI images thenunderwent correction for bias field inhomogeneities[161] and eddy current distortions. Using FMRIB’s Diffusion Toolbox from FMRIB’s Software Library (FSL),
diffusion tensor models were assigned to each voxel togenerate DTI parametric maps: fractional anisotropy(FA), axial (AxD), mean (MD), radial (RD) diffusivity. In
the T2WI scans, bias field inhomogeneities were corrected and quantitative T2 relaxation maps were generated with JIM software (RRID: SCR_009589, Xinapse
Systems Ltd; West Bergholt, Essex; United Kingdom). A
modified Australian Mouse Brain Mapping Consortium(AMBMC) atlas with 40 bilateral regions (Ullman et al.,2013, Richards et al., 2011) was non-linearly registeredto each scan and regional labels were applied with theAdvanced Normalization Tools (ANTs). The atlas overlay
was manually inspected, and misalignments were corrected with ITK-SNAP. Regional metric outliers withineach group were excluded with 1.5xIQR above the firstquartile or below the third quartile.
To assess the effects of the various treatments on structural connectivity, DTI scans were reconstructed on DSI
studio with a diffusion sampling length ratio of 0.85 (Jan25, 2022, build; http://dsi-studio.labsolver.org/). Wholebrain seeding was performed and manually inspected
for tract quality. The AMBMC region designations werethen used to generate structural connectivity matrices.
Groups were averaged and plotted with Circos (http://
circos.ca/ [162]), . Connectivity symmetry was evaluated using an Asymmetry Index ([Left-Right/Left + Right]
×100%). For tractography, DTI scans were reconstructedusing a diffusion sampling length ratio of 0.75 for connections between site of injury and anterior cingulate cortex(ACC). The seed was manually delineated in the cortex
above the dorsal hippocampus and the target ACC region
was generated from the AMBMC atlas. Deterministic
tractography was performed using an angular threshold of 70, a step size of 0.06, and 0.80 smoothing. Tractconfirmation utilized viral tracing experiments from the
Allen Brain Institute (https://connectivity.brain-map.
org/) where our tracts closely matched those of experiment #126,907,302.

Iannucci et al. Journal of Neuroinflammation(2024) 21:165

Statistical analysis

Power analysis was done using GPower Software (Version2.0) to determine necessary samples sizes and found thatn = 12 was sufficient to achieve β = 0.836 in our neurobehavioral assays, also allowing for sufficient sample sizes
for subsequent IHC and flow analyses. Due to limitations
encountered when running the study, the actual sample
size for vehicle-treated groups was n = 6 for neurobehavioral assays. While low, this sample size still allowed for
effect size d = 0.94 in the Barnes Maze for WT Sham + Veh
vs. 5xFAD Sham + Veh. Subsequent statistical analysis
was carried out using both GraphPad Prism (Version
9.0) and R. For analysis of WT Sham + Veh versus 5xFAD
Sham + Veh, a student’s t-test was used to assess genotype
differences. Further analysis of the effects of CAP and FPI
on the 5xFAD mice for flow cytometry, behavior, immunohistochemistry, and vessel painting data were analyzed
by one-way analysis of variance (ANOVA) with post-hoc
testing using the Holm-Sidak correction for planned
comparisons. For digigait, the means and standard deviation for each day for each metric were calculated using
Python, and a student’s t-test was used to assess differences in pre-planned group comparisons. Generalized
estimating equation (GEE) models were used for regression modeling of the burrowing and social interaction
longitudinal variables, with natural cubic splines forrepresenting time courses as smooth functions [163].
In the GEE models, interactions were included between
the time course curves, surgery (FPI / Sham), and drug
(CAP / Vehicle). Tests were conducted for both surgery
and drug effects in terms of overall differences between
mean time course curves via F-tests of nested models.
For PST, Fisher’s Exact Test was used to test for differences in proportions of mice that visited novel objects.
Additional exploratory statistical analysis was completedin R to investigate correlations between pathological endpoints and behavioral data via. For all statistical testing,significance was considered p < 0.05 and a trend was considered 0.05 < p < 0.10.
Abbreviations
ACC	Anterior Cingulate Cortex
AD	Alzheimer’s disease
ANOVA	one-way analysis of variance
AxD	axial diffusivity
CAP	CLIP antagonist peptide
CLIP	Major Histocompatibility Complex class II-associated invariant
peptideDCX	doublecortin
DG	dentate gyrus
DiI	1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate
DMSO	dimethyl sulfoxide
DPI	days post injury
DSM	Diagnostic and Statistical Manual of Mental Disorders
DTI	diffusion tensor imaging
EPM	elevated plus mazeFA	fractional anisotropy
FBS	fetal bovine serum
FPI	fluid percussion injury

Page 20 of 24

GEE	generalized estimating equationi.p.	intraperitoneal
MCI	mild cognitive impairment
MD	mean diffusivity
MHCII	Major Histocompatibility Complex class II
MRI	magnetic resonance imaging
PBS	phosphate buffered saline
PFA	paraformaldehyde
PST	pattern separation testRD	radial diffusivity
RSC	Retrosplenial Cortex
SNP	sodium nitroprusside
TBI	Traumatic brain injury
WT	Wild type

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03146-z.
Supplementary Material 1
Supplementary Material 2
Supplementary Material 3
Supplementary Material 4
Acknowledgements
The mouse strain used for this research project, B6.Cg-Tg(APPSwFlLon,
PSEN1*M146L*L286V)6799Vas/Mmjax, RRID: MMRRC_034848-JAX, wasobtained from the Mutant Mouse Resource and Research Center (MMRRC) at
The Jackson Laboratory, an NIH-funded strain repository, and was donatedto the MMRRC by Robert Vassar, Ph.D., Northwestern University. The authors
would like to acknowledge Victoria Arismendi, Kassandra Pulido, Ashley
R. Taylor, and Roberto Jimenez for their assistance running behavioral
experiments. The diagram in Fig. 1 was created using Biorender.com.
Author contributions
JI: Conceptualization, Methodology, Formal Analysis, Investigation, Writing –Original Draft, Writing – Review & Editing RD: Investigation SB: Investigation
EMA: Investigation BPN: Methodology, Formal Analysis AJ: Investigation,
Formal Analysis MK: Methodology, Formal Analysis RPT: Formal Analysis
AP: Formal Analysis, Data Curation SB: Investigation LV: Investigation KAS:
Methodology, Formal Analysis, Resources DCJ: Formal Analysis, Data
Curation AD: Formal Analysis, Data Curation, Writing – Review & Editing AO:
Methodology, Formal Analysis, Resources, Supervision, Writing – Review& Editing MKNR: Conceptualization, Methodology, Resources, Writing –Review & Editing, Supervision, Funding Acquisition LAS: Conceptualization,
Methodology, Resources, Writing – Original Draft, Writing – Review & Editing,
Supervision, Project Administration, Funding Acquisition. All authors read andapproved the final manuscript.
Funding
This work was supported by National Institutes of Health (NIH) RO1NS104282
and by an Administrative Supplement to NIH RO1NS104282S1. The work isalso supported by the Department of Defense (DoD), USA, Peer Reviewed
Alzheimer’s Research Program (PRARP) grant #W81XWH2210690 and#W81XWH2210280. The views, opinions and/or findings contained in thisreport are those of the author(s) and should not be construed as an official
Department of the Army Position, policy or decision unless so designated byother documentation. This work is also supported by Alzheimer’s Association
Research Fellowship (AARF) #AARF-23-1144965.
Data availability
The datasets generated during and/or analyzed during the current study areavailable from the corresponding author on reasonable request.

Iannucci et al. Journal of Neuroinflammation(2024) 21:165

Declarations
Ethics approval
All work was approved by the Texas A&M Institute for Animal Care and Use
Committee (IACUC) under animal use protocol #2020 − 0140.
Consent for publication
Not applicable.
Competing interests
M. Karen Newell-Rogers and Richard P. Tobin are inventors on patents related
to CAP. None of the other authors have any competing interests in this work.
Author details
1
Department of Neuroscience and Experimental Therapeutics, College ofMedicine, Texas A&M University, Bryan, TX, USA
2
Department of Medical Physiology, College of Medicine, Texas A&M
University, Bryan, TX, USA
3
Division of Biomedical Sciences, University of California Riverside,
Riverside, CA, USA
4
Department of Surgery, Division of Surgical Oncology, University ofColorado Anschutz Medical Campus, Denver, CO, USA
5
Department of Biostatistics and Data Science, Department ofOrthopaedics and Rehabilitation, The University of Texas Medical Branch,
Galveston, TX, USA
6
Department of Statistics, College of Arts & Sciences, Texas A&M
University, College Station, TX, USA
Received: 19 January 2024 / Accepted: 29 May 2024

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