A novel model of trauma-induced cerebellar injury and myelin loss in mouse organotypic cerebellar slice cultures using live imaging

Accepted Manuscript Title: A novel model of trauma-induced cerebellar injury and myelin loss in mouse organotypic cerebellar slice cultures using live imaging Authors: Gemma Llufriu-Dab´en, Delphine Meffre, Charbel Massaad, Mehrnaz Jafarian-Tehrani PII: DOI: Reference:

S0165-0270(18)30290-5 https://doi.org/10.1016/j.jneumeth.2018.09.023 NSM 8125

To appear in:

Journal of Neuroscience Methods

Received date: Revised date: Accepted date:

2-5-2018 13-9-2018 18-9-2018

Please cite this article as: Llufriu-Dab´en G, Meffre D, Massaad C, Jafarian-Tehrani M, A novel model of trauma-induced cerebellar injury and myelin loss in mouse organotypic cerebellar slice cultures using live imaging, Journal of Neuroscience Methods (2018), https://doi.org/10.1016/j.jneumeth.2018.09.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A novel model of trauma-induced cerebellar injury and myelin loss in mouse organotypic cerebellar slice cultures using live imaging Gemma Llufriu-Dabén, Delphine Meffre, Charbel Massaad, Mehrnaz Jafarian-Tehrani INSERM UMR-S 1124, Paris Descartes University, Sorbonne Paris Cité, Faculté des Sciences

IP T

Fondamentales et Biomédicales, 45 rue des Saints-Pères, 75006 Paris, FRANCE Corresponding author :

SC R

Mehrnaz Jafarian-Tehrani

A

45 rue des Saints-Pères, 75006 Paris, FRANCE

TE D

M

Phone: (+ 33) 01 70 64 99 76 [email protected]

N

Faculté des Sciences Fondamentales et Biomédicales,

U

INSERM UMR-S 1124, Paris Descartes University, Sorbonne Paris Cité,

Author names, affiliations:

Gemma Llufriu-Dabén, PharmD, PhD

EP

INSERM UMR-S 1124, Paris Descartes University, Sorbonne Paris Cité,

CC

45, rue des Saints-Pères, 75006, Paris, France Telephone: (+33) 01 42 86 41 61

A

Fax: (+33) 01 42 86 38 68 Email: [email protected] Delphine Meffre, Associate Professor, PhD INSERM UMR-S 1124, Paris Descartes University, Sorbonne Paris Cité,

2

45, rue des Saints-Pères, 75006, Paris, France Telephone: (+33) 01 42 86 41 61 Fax: (+33) 01 42 86 38 68 Email: [email protected]

INSERM UMR-S 1124, Paris Descartes University, Sorbonne Paris Cité,

SC R

45, rue des Saints-Pères, 75006, Paris, France

IP T

Charbel Massaad, Full Professor, PhD

Telephone: (+33) 01 70 64 99 76

U

Fax: (+33) 01 42 86 38 68

A

N

Email: [email protected]

M

Mehrnaz Jafarian-Tehrani, Full Professor, PhD

INSERM UMR-S 1124, Paris Descartes University, Sorbonne Paris Cité,

TE D

45, rue des Saints-Pères, 75006, Paris, France Telephone: (+33) 01 70 64 99 76

EP

Fax: (+33) 01 42 86 38 68

Email: [email protected]

CC

INSERM UMR-S 1124, Paris Descartes University, Sorbonne Paris Cité, Faculté des Sciences

A

Fondamentales et Biomédicales, 45 rue des Saints-Pères, 75006 Paris, FRANCE Corresponding author : Delphine Meffre, Associate Professor, PhD Charbel Massaad, Full Professor, PhD Mehrnaz Jafarian-Tehrani, Full Professor, PhD

3

A

CC

EP

TE D

M

A

N

U

SC R

IP T

Email: [email protected]

4

Highlights A novel weight-drop model of TBI in mouse cerebellar slice cultures



Live imaging of trauma-induced cell death and myelin loss ex vivo



Etazolate protective effects on cerebellar injury and myelin loss ex vivo



Suitable model to test active compounds against cerebellar injury

A

CC

EP

TE D

M

A

N

U

SC R

IP T



5

Abstract Background: Traumatic brain injury (TBI) induces significant cognitive deficits correlated with white matter injury (WMI), involving both axonal and myelin damage. Several models of TBI ex vivo are available to mimic focal impact on brain tissue. However, none of them addressed the study of trauma-induced myelin damage.

IP T

New method: The aim of this study was to set up a novel ex vivo weight-drop model on organotypic cultures obtained from mouse cerebellum, a highly myelinated structure, in order

SC R

to study the temporal evolution of cerebellar lesion and demyelination. The extent of injury was measured by propidium iodide (PI) fluorescence and demyelination was evaluated by loss of GFP-fluorescence in cerebellar slices from PLP-eGFP mice.

U

Results: Live imaging of slices showed an increase of PI-fluorescence and a significant loss of

N

GFP-fluorescence at 6h, 24h and 72h post-injury. At the impact site, we observed a loss of

A

Purkinje cells and myelin sheaths with a marked loss of myelin protein MBP at 72h following

M

injury. Etazolate, a known protective compound, was able to reduce both the PI-fluorescence increase and the loss of GFP-fluorescence, emphasizing its protective effect on myelin loss.

TE D

Comparison with existing methods and conclusions: In line with the existing models of focal injury, we characterized trauma-induced cerebellar lesion with an increase of PI fluorescence by live imaging. Our findings describe a novel tool to study trauma-induced myelin damage in

EP

cerebellar slices and to test biomolecules of therapeutic interest for myelin protection.

CC

Keywords: traumatic brain injury; focal lesion; demyelination; cerebellum; neuroprotection;

A

myelin repair

6

Introduction Traumatic brain injury (TBI) is a critical public health and socio-economic problem in both industrialized and developing countries, causing death and disability, especially among young adults (Maas et al., 2017; Majdan et al., 2016). TBI is the result of a direct or indirect damage to the brain induced by mechanical forces of compression, stretching, shearing or

IP T

tearing, resulting in haemorrhage, contusions, axonal injury and lacerations (Adams et al., 1983; Saatman et al., 2008). Trauma initiates a cascade of secondary pathophysiological

SC R

events, such as excitotoxicity, neuroinflammation, and oxidative stress that worsen brain

pathology from hours to days following the mechanical insult (Morganti-Kossmann et al., 2007; Schmidt et al., 2005). Apoptotic and necrotic neurons have been observed within contusions

U

and in regions far from the lesion site in the days and weeks after TBI, while degenerating

N

oligodendrocytes and astrocytes have been identified within injured white matter tracts

A

(Raghupathi, 2004). The damage of both axons and myelin sheaths has been reported in

M

head-injured patients and in TBI animal models (Armstrong et al., 2015a; Flygt et al., 2018; Herrera et al., 2016; Rodriguez-Grande et al., 2018; Taib et al., 2017). In addition, it has been

TE D

shown that myelin abnormalities correlate with cognitive and motor deficits after TBI (Croall et al., 2014; Kinnunen et al., 2011). However, it remains to be elucidated whether trauma-induced demyelination is a primary or secondary event, caused by axonal degeneration and/or

EP

neuroinflammation (Johnson et al., 2013; Taib et al., 2017). Furthermore, oligodendrocyte cell death after traumatic injury has been also reported in both post-mortem human brains (Shaw

CC

et al., 2001) and in experimental models of focal TBI (Dent et al., 2015; Flygt et al., 2013; Lotocki et al., 2011) and could contribute to demyelination of intact axons (Armstrong et al.,

A

2015a). In this context, several studies underline the importance of white matter integrity and propose demyelination as a new therapeutic target to enhance neurological recovery after TBI (Armstrong et al., 2015a, 2015b; Shi et al., 2015). Therefore, model systems that allow rapid screening of myelin damage and repair are needed. The aim of this study was to develop a novel weight-drop model of cerebellar injury

7

and myelin loss based on an organotypic slice culture of the cerebellum. The extensive myelination in the cerebellum (Notterpek et al., 1993) makes it a suitable structure for studying the fate of white matter after injury. Culture of cerebellar slices is the most commonly used organotypic culture to study myelination and remyelination processes (Doussau et al., 2017), while organotypic culture of hippocampal slices is used to investigate the neuroprotective

IP T

effects of drugs and to assess neurogenesis. The major novelty of our study was to present a new model allowing the live imaging of myelin loss after traumatic injury in cerebellar slice culture. Although clinical evidence and animal models of TBI show that cerebellum is often

SC R

indirectly affected (Park et al., 2007; Spanos et al., 2007; Staffa et al., 2012; Wang et al., 2016),

research findings on cerebellar injury remain sparse. Few reports described in vivo models of

U

traumatic cerebellar injury (Potts et al., 2009), while ex vivo model systems using cerebellum are lacking (Morrison et al., 2011). Thus, our goal was to study the trauma-induced cerebellar

N

injury and demyelination by live imaging of cerebellar slices obtained from wild type C57BL/6

A

and transgenic PLP-eGFP mice. The latter has an advantage to overexpress an enhanced

M

green fluorescent protein (eGFP) in the oligodendroglial lineage, driven by the mouse myelin

TE D

proteolipid protein (PLP) gene promoter (Mallon et al., 2002). In order to evaluate cerebellar injury and myelin loss, we performed live imaging of cerebellar slices at different time points post-injury (6h, 24h, 72h) to assess the time course of cell death and myelin loss by propidium

EP

iodide (PI) incorporation and eGFP fluorescence intensity, respectively. The usefulness of such models is also to test the pharmacological compounds with

CC

potential therapeutic activity on cerebellar injury and myelin protection/repair. To validate our model, we used etazolate, a pyrazolopyridine derivative, shown to be an α-secretase activator

A

with neuroprotective and remyelinating effects in vitro and in vivo (Llufriu-Dabén et al., 2018; Marcade et al., 2008; Siopi et al., 2013). In previous studies, Marcade and collaborators have shown that etazolate, at low micromolar ranges, is able to protect neurons against A β neurotoxicity by enhancing the release of a soluble neuroprotective protein, sAPPα (Marcade et al., 2008), through α-secretase cleavage of amyloid precursor protein (APP) (Nalivaeva and

8

Turner, 2013). Our group has also shown that etazolate promotes neuroprotection and improves histological and functional outcomes in a model of closed-head injury in vivo (Siopi et al., 2013). Recently, we have also shown that etazolate is protective of myelin sheaths and exerts remyelinating effects after chemical-induced demyelination ex vivo and in vivo (LlufriuDabén et al., 2018).

IP T

In this report, live imaging of slice cultures clearly showed trauma-induced myelin damage in the cerebellar slices and the protective effect of etazolate providing a new model to

A

CC

EP

TE D

M

A

N

U

SC R

study cerebellar injury and myelin loss.

9

Materials and Methods Animals Organotypic culture of cerebellar slices was performed on mice using either C57BL/6 (Janvier, Le Genet St Isle, France) or transgenic PLP-eGFP mouse pups (generated by Dr. W.B. Macklin, Cleveland Clinic Foundation, Ohio, USA). Animals were housed in a controlled

IP T

temperature environment (22 ± 2°C), under a 12h light/dark cycle. Animal care and all experiments were approved by the Paris Descartes University Animal Ethics Committee

SC R

(CEEA34.MJT.075.12), respecting the French regulations and the European Communities Council Directive of September 2010/63/UE, on the protection of animals used for scientific purposes.

U

Organotypic culture of cerebellar slices

N

Cerebellar slices were obtained from post-natal day 8-10 (P8-P10) mouse pups. Brains

A

were first removed and placed in a cold dissection medium containing Phosphate Buffer Saline

M

(PBS, 0.1M) supplemented with 5 mg/mL D-glucose (Sigma-Aldrich, Lyon, France). After removing the meninges, the cerebellum was dissected and 350 m-thick parasagittal slices

TE D

were cut using a tissue chopper (McIlwain Tissue Chopper, UK) in a sterile environment. Slices isolated from the vermis region (4-6 per membrane) were then transferred onto membranes of

EP

30 mm Millipore culture inserts with 0.4 m pore size (Millipore, Molsheim, France). The inserts were placed in 6-well tissue culture plates with 1 mL of medium containing 50% Basal Medium

CC

with Earle’s salts (BME) (Invitrogen, Gaithersburg, MD), 2.5% Hank’s Balanced Salt Solution (HBSS) (Life Technologies, Grand Island, NY), 25% heat-inactivated horse serum (Thermo

A

scientific, Courtaboeuf, France), 1% glutaMAX® (Thermo scientific, Courtaboeuf, France), 5 mg/mL D-glucose (Sigma-Aldrich, Lyon, France) and penicillin (0.1 U/mL) - streptomycin (0.1 µg/mL). Slices were maintained at 35°C in a humidified atmosphere of 5% CO2 and the medium was changed every 3-4 days. Ex vivo traumatic cerebellar injury

10

At 7 days of culture in vitro (DIV), the medium was changed to an experimental medium immediately prior to the induction of mechanical injury. The experimental medium was serumfree and consisted of 75% BME, 2.5% HBSS, 1% glutaMAX®, 5 mg/mL D-glucose, penicillin (0.1 U/mL) - streptomycin (0.1 µg/mL) and 5 g/mL propidium iodide (PI) (Sigma-Aldrich, Lyon, France). Etazolate (Tocris Bioscience, Lille, France), at the neuroprotective and remyelinating

IP T

concentration of 2 M (Llufriu-Dabén et al., 2018; Marcade et al., 2008), or its vehicle (PBS, 0.1M) were added in the experimental medium.

SC R

Mechanical trauma was performed using a weight-drop injury model, adapted from previous

reports (Adamchik et al., 2000; Adembri et al., 2004; Coburn et al., 2008) on the cerebellar tissue. A specially designed apparatus (Fig. 1) was constructed and pilot experiments were

U

performed to induce injury without severe tissue damage. A steel stylus of 324 mg weight and

N

of 1 mm diameter was dropped on the cerebellar slice from 5 mm, 7 mm or 10 mm height for

A

a 15 second contact time at 7 DIV. The impact focus was located on the largest lobule of each

M

cerebellar slice. Smooth distal part of the stylus excluded any tissue perforation. In each set of experiments, we used 5 to 15 slices obtained from at least 2 different animals for a given group

TE D

and we have performed three to seven independent experimental settings. Finally, cultures returned to the incubator until evaluation at different time points up to 72h post-injury (Fig. 2). Assessment of cerebellar injury and demyelination following traumatic injury by live imaging of

EP

PI incorporation and GFP fluorescent intensity

CC

Live imaging was performed using an inverted epifluorescence microscope (Zeiss Axio Observer Z1 Inverted Microscope) with a low-power objective (10x/NA0.3; Zeiss EC-Plan-

A

NEOFLUO) able to visualize red and green fluorescence. During live imaging, slices were kept at 35°C in a humidified atmosphere of 5% CO2. Images were captured with a digital camera (ORCA-flash20.8-11440-10C). Prior to the impact injury, slices were imaged for PI fluorescence, and non-viable PI+ slices with more than negligible baseline fluorescence were excluded from the study. Each slice was imaged to evaluate the time course of cerebellar lesion and demyelination. In addition, each slice was imaged 1h before the injury (only for PLP11

eGFP slices), and 6, 24 and 72h post-injury (for all slices) (Fig. 2). A single image of one slice is composed of 30 images as mosaic performed using ZEN 2012 software (ZEN 2012 blue edition, Carl Zeiss Microscopy GmbH). Images were then converted to TIFF format and the distribution of intensities was plotted as a histogram (0-256 grey levels). Only the red and green channels were recorded for the quantification of PI- and GFP-fluorescence, respectively.

IP T

The extent of cerebellar injury was assessed in organotypic cerebellar slices obtained from C57BL/6 mice using PI, a highly polar dye that only penetrates cells with damaged

SC R

membranes, as previously described (Adamchik et al., 2000; Adembri et al., 2004; Coburn et

al., 2008). Inside the cell, PI binds to DNA and RNA (Rieger et al., 2011) and becomes fluorescent, with a peak emission in the red region of visible spectrum. In order to assess the

U

intensity of PI fluorescence as an indicator of cerebellar injury, the number of pixels was

N

calculated from the whole slice area to take into consideration the eventual cell death out of

A

the lesion area. Therefore, we used the ImageJ software (1.46r, NIH, USA) to delineate the

M

whole slice area. Data were expressed as total number of pixels transcending the threshold of 35 levels. The latter was determined under our experimental conditions in order to reduce the

TE D

level of background signal and optimize the quantitative analysis. The increase in the number of pixels in response to injury was used as an indicator of the extent of lesion. Slices from PLP-eGFP mice were used for live imaging of demyelination post-injury. Each slice

EP

was imaged over time up to 72h (Fig. 2). The PLP-eGFP slices were traumatized and an area

CC

into the injury zone, beforehand spotted by PI, was delimited using the ImageJ software (1.46r, NIH, USA). For control slices, a matched area was determined. The loss of GFP intensity was considered as an indicator of demyelination. Data were expressed as the percentage of GFP-

A

fluorescence emitted at each time point reported at GFP-fluorescence emitted before injury, for each slice independently. Immunohistochemistry

12

At 72h post-injury, a set of slices obtained from C57BL/6 mice were fixed with 4% paraformaldehyde (PFA) solution for 40 minutes at room temperature (RT) and stocked at 4°C until immunolabelling (Fig. 2). Fixed slices were washed and then blocked with L-lysine (0.1M) in a PBS medium buffer containing 0.25% triton-X, 0.2% gelatine, 0.1% sodium azide (PBSGTA) for 1h at RT. Slices were then incubated overnight at 4°C with primary antibodies anti-

IP T

MAG for myelin sheaths (1/1000; MAB1567, Millipore, Molsheim, France) and anti-Calbindin for Purkinje cells (1/10 000; CB38a, Swant, Marly, Switzerland) carried out in a PBS-GTA medium buffer. The following day, slices were washed and incubated with secondary

SC R

antibodies: goat anti-mouse antibody coupled to Alexa-488 (1/1000; A21121, Thermo

scientific, Courtaboeuf, France) and a goat anti-rabbit coupled to Alexa-405 (1/500; A31556,

U

Thermo scientific, Courtaboeuf, France), for 2h at RT. The slices were finally mounted with Fluoromount (Southern Biotech, Birmingham, USA). Images were taken using a confocal

A

N

microscope LSM 710 (Carl Zeiss Inc.) with 20x lens (Zeiss Plan-APOCHROMAT 20x/0.8).

M

Western Blotting of myelin proteins

For each experiment, we prepared a pool of samples obtained from 5 to 8 cerebellar

TE D

slice cultures per condition (whole slice for control condition, and a 1.5 mm-diameter-punch of the injured area at 72h post-injury) (Fig. 2). Proteins were extracted in cold RIPA (RadioImmuno Precipitation Assay) buffer (50 mM Tris HCl, pH 7.5; 150 mM NaCl; 0.1% SDS

EP

(Sodium Dodecyl Sulfate); 0.5% sodium deoxycholate; 1% NP-40; 10 mM sodium

CC

pyrophosphatase; 0.1 mM sodium fluorate; Ethylenediaminetetraacetic acid (EDTA) 5 mM; protease inhibitor (cOmplete, Mini, EDTA-free, Roche, Boulogne-Billancourt, France) and phosphatase inhibitor (Protease Inhibitor Cocktail 2, P5726, Sigma-Aldrich, Lyon, France).

A

Protein content was determined using the ReduCing agent and Detergent Compatible protein assay (RC-DC protein assay kit, BioRad, Hercules, California, USA) with BSA (Bovine Serum Albumin) as standard. Aliquots of 30 g of protein for each condition were separated on 10% SDS-polyacrylamide gels by electrophoresis and blotted onto PVDF (polyvinylidene difluoride) membranes. Non specific binding sites on the transblots were blocked with 5% BSA diluted in

13

Tris Buffered Saline with 0.1% Tween20 (TBST) for 1h at RT. Membranes were then incubated overnight at 4°C with primary antibodies diluted in 5% BSA-TBST: -actin (1/10 000; ab8227, Abcam, Cambridge, UK), and MBP (1/200; MAB381, Millipore, Molsheim, France). After several washes with TBST, the membranes were incubated for 2h with the corresponding secondary antibody diluted in 5% BSA-TBST: goat anti-rabbit (1/20 000; 111-035-144;

IP T

Jackson, Cambridge, UK) and goat anti-mouse (1/20 000; 170-5047-MSDS; BioRad Hercules, California, USA), both coupled to HRP. Following several washes, membranes were then

revealed with the Pierce ECL 2 western blotting substrate kit (Thermo Scientific, Courtaboeuf,

SC R

France) and imaged with ImageQuant LAS 4000 (GE Healthcare, Aulnay sous bois, France). Statistical analysis

U

Data were expressed as mean  standard error of the mean (s.e.m). We defined n as

N

a number of slices for a given group obtained from three to seven experimental settings. For

A

our data analysis, we did not consider cerebellar slices obtained from the same animal as

M

replicates because of the complex structure of cerebellum (Apps and Hawkes, 2009; Reeber et al., 2013). The data were analysed using GraphPad Prism statistical software (Prism v4,

TE D

GraphPad, La Jolla, CA). We used the non-parametric Kruskal-Wallis test for multiple group comparison followed by Mann-Whitney for comparison of two data sets. Differences with a P

A

CC

EP

value under 0.05 were considered to be statistically different.

14

Results Temporal evolution of trauma-induced cerebellar injury by live imaging of PI incorporation In order to obtain an ex vivo model of cerebellar injury, we have used a weight-drop injury model and designed a device to induce reproducible injury without severe tissue damage in cerebellar slices. Control slices incubated with PI displayed very low levels of fluorescence

IP T

(Fig. 3A, 3B), whereas quantitative analysis of slices subjected to impacts of 5 mm, 7 mm or 10 mm height, revealed a significant increase of PI fluorescence at 6h, 24h, 72h post-injury

SC R

compared to control slices (Table 1). The weight-drop falls produced a definite area of damage

in cerebellar slices, with the lowest variability in PI-fluorescence for the height of 7 mm (Table 1). Hence the weight-drop height of 7 mm was selected for all subsequent experiments.

U

Trauma-induced cerebellar injury following a weight-drop fall of 7 mm was visualized by live

N

imaging of PI staining on the entire slice area. Each slice has been imaged three times (6h,

A

24h, 72h) to study the temporal evolution of trauma-induced injury. Six hours after trauma, the

M

PI-fluorescence was significant at the lesion area of vehicle-treated injured slices. The PIfluorescence continued to significantly increase from 24h to 72h (P<0.0001) (Fig. 3A, 3B).

TE D

Treatment of cerebellar slices with etazolate reduced the PI staining at all time points postinjury (P=0.054 at 6h; P<0.01 at 24h; P<0.01 at 72h) (Fig. 3A, 3B). Exposure of control slices to etazolate did not induce any significant increase of PI-fluorescence at any time point studied,

EP

as compared to vehicle-treated control slices (Fig. 3A, 3B).

CC

Qualitative analyses of Purkinje cells and myelin sheaths following traumatic injury Next, we investigated the effect of trauma on Purkinje cells and myelin sheaths in

A

cerebellar slices at 72h post-injury. Confocal images of MAG+ myelin sheaths and calbindin+ Purkinje cell body and axons highlighted loss of staining within the injured area (Fig. 4A). Interestingly, the double MAG/CaBP immunolabeling was preserved in etazolate-treated injured slices (Fig. 4A). Control slices treated with etazolate did not show any modification of both markers compared to vehicle-treated control slices (Fig. 4A). Western blot analysis showed a decrease in the amounts of MBP (21 and 18 KDa isoforms), a specific myelin protein, 15

within the injured area at 72h post injury (Fig. 4B), which was reversed by etazolate treatment (Fig. 4B). The quantitative analysis of MBP blots did not show any significant difference between groups (Fig. 4C) due to the high variability across the blots and a small sample size. However, we observed a reproducible decrease of MBP after injury, and also MBP recovery after etazolate treatment in each independent experiment.

IP T

Temporal evolution of demyelination in PLP-eGFP cerebellar slices following traumatic injury by live imaging of GFP fluorescent intensity

SC R

The effect of trauma on myelin sheaths was investigated by live imaging of each

cerebellar slice. In order to achieve a quantitative analysis of trauma-induced demyelination, cerebellar slices from PLP-eGFP mice were used. In these mice, the myelin sheaths and

U

oligodendrocytes are highlighted in green (Fig. 5A). The GFP-fluorescence intensity was

N

measured over time for each slice: before the injury, and at 6h, 24h and 72h post-injury. Thus,

A

live imaging of GFP allowed us to perform a time course study of myelin loss in a given slice

M

over time, providing an easy tool for a quantitative evaluation of demyelination post-injury. The use of PLP-eGFP mice in our study provides a reproducible analysis of fluorescence intensity

TE D

and has an advantage over the immunostaining of myelin proteins on thick slices, which present more variability in antibody staining due to permeability issues. Trauma caused a reduction of GFP-fluorescence within the injured area (PI+), by 30-35% at 6h, 24h and 72h

EP

after injury (P<0.0001), indicating significant myelin loss (Fig. 5A, 5B). Interestingly, etazolate

CC

treatment increased GFP-fluorescence by 20% in injured slices at all time points post-injury, compared to vehicle-treated injured slices (P<0.05 at 6h; P=0.054 at 24h; P=0.053 at 72h) (Fig. 5A, 5B). Exposure of control slices to etazolate did not induce any significant modification

A

of GFP-fluorescence at any time point studied (Fig. 5A, 5B).

16

Discussion This report describes a novel ex vivo model of cerebellar injury using organotypic cerebellar slice cultures that could be used as a new tool to study trauma-induced cerebellar injury and myelin loss. It is noteworthy that approaches targeting demyelination after traumatic CNS injuries

IP T

received increased attention (Kondiles and Horner, 2018; Scheller et al., 2017; Shi et al., 2015; Weber et al., 2018). Despite the numerous clinical trials undertaken so far, no

SC R

neuroprotective/glioprotective therapy is yet available against TBI (Diaz-Arrastia et al., 2014). Although a few reports described some promise for white matter integrity, especially in studies of traumatically induced axonal injury (Cross et al., 2015; Singleton et al., 2001; Wang et al.,

U

2015), therapeutic strategies enhancing myelin sheath protection and repair following TBI are

N

still lacking. Thus, among other challenges, there is also a need to develop model systems for

A

trauma-induced injury and myelin loss that allow rapid screening of novel pharmacological

M

compounds in vitro or ex vivo (Sundstrom et al., 2005).

In this report, we developed a novel model of cerebellar injury and myelin loss, since

TE D

the highly myelinated structure of the cerebellum is suitable to study the fate of myelin after injury (Notterpek et al., 1993). Furthermore, organotypic culture of tissue slices offers some advantages over dissociated cell culture because the former mimics the in vivo state of tissue,

EP

and maintain the architectural connexions between the heterogeneous cell populations,

CC

allowing an easier and faster access to the tissue (Morrison et al., 2011). Several ex vivo models of TBI have been previously described, produced by different

A

mechanical insults as compression (weight-drop models), acceleration, stretch and shear, all relevant forces occurred in head-injured patients or animal models of TBI (Kumaria and Tolias, 2008; Miller et al., 2017, 2015; Morrison et al., 2011). Our trauma device aimed to produce a focal lesion in the cerebellum, highlighted by PI-fluorescence. The PI-fluorescence was already evident in the acute phase of the injury at 6h, evolving at 24h and 72h after injury suggesting the maturation of trauma-induced tissue lesion also reported in vivo (Flierl et al., 2009). In

17

addition, the exacerbation of the lesion over time in our model is similar to the lesion pattern found in other ex vivo weight-drop injury models in the hippocampus (Adembri et al., 2004; Coburn et al., 2008; Fahlenkamp et al., 2011), the most studied structure ex vivo (Morrison et al., 2011). Compared to previous reports, the energy of the impact applied to induce injury in organotypic cerebellar slice culture was higher than the one described for injured hippocampal

IP T

slices, but lower for the injured spinal cord slices (Adamchik et al., 2000; Krassioukov et al., 2002). In fact, the vulnerability of CNS tissue to injury is structure-dependent, the hippocampus

SC R

is being more vulnerable to injury (Bartsch et al., 2015; Wang and Michaelis, 2010).

In parallel, the immunohistochemical analyses performed at 72h post-injury showed a loss of Purkinje cells as well as myelin loss within the injured area. Thereafter, the assessment

U

of the specific myelin proteins such as MBP by Western blotting at 72h after injury showed a

N

decrease of the amount of MBP within the injured area. The major novelty of this work is that

A

our model presents a new tool allowing the live imaging of myelin loss after the traumatic insult

M

on PLP-eGFP cerebellar slices. As expected, our results showed a marked demyelination, highlighted by loss of GFP intensity from 6h to 72h post-injury. Studies regarding ex vivo

TE D

trauma-induced demyelination are very rare. One report describes an in vitro stretch model of TBI on primary co-cultures of neurons and oligodendrocytes, showing that unmyelinated axons are more vulnerable to stretch-injury than myelinated axons (Staal and Vickers, 2011).

EP

In this report, we also studied the effect of a pyrazolopyridine derivate, etazolate, on

CC

trauma-induced cerebellar injury and demyelination. Interestingly, injured slices treated with etazolate presented an improvement of both Purkinje cells and myelin sheaths, with an increase of the amount of MBP protein. Etazolate was able to protect myelin sheaths,

A

manifested by an increase of the GFP intensity post-injury. Moreover, the cerebellar lesion, highlighted by an increase of PI incorporation, was reduced by etazolate treatment after injury. It is noteworthy that the extent of etazolate induced-recovery was different regarding the methodology used, since each assessment provides different and complementary information (global tissue injury by PI analysis, myelin integrity by GFP analysis, and MBP protein by WB

18

analysis). In addition, further investigations are required to characterize the PI+ cell type in the injured cerebellar slices. In fact, besides neurons and oligodendrocytes, other cell types in the cerebellum could be also affected after traumatic injury. Overall, our present data including our previous reports provide evidence that etazolate is beneficial in cerebral lesions in vivo (LlufriuDabén et al., 2018; Siopi et al., 2013) and ex vivo (Llufriu-Dabén et al., 2018), suggesting that

IP T

the same pathways underlying the lesion might be involved in these models. In fact, our previous study showed for the first time the neuroprotective effect of etazolate, as an αsecretase activator, in a weight-drop model of TBI in mice (Siopi et al., 2013). Our current study

SC R

highlights the protective effect of etazolate in another new structure, the cerebellum, and emphasizes its protective effect on myelin sheaths after traumatic injury. Thus, the beneficial

U

effect of etazolate observed in this study could be due to both neuroprotective and myeloprotective mechanisms exerted concomitantly. Moreover, we cannot exclude an additional

N

remyelinating effect of etazolate in our cultures. Indeed, the beneficial effect of etazolate on

A

myelin loss observed in this study is in line with our recent findings highlighting both myelo-

M

protective and remyelinating effect of etazolate, as an an α-secretase activator, after

TE D

demyelination (Llufriu-Dabén et al., 2018). However, other action mechanism of etazolate should not be ruled out. In fact, etazolate acts as an agonist of adenosine receptor (Daly et al., 1988) and a modulator of GABAA (Thompson et al., 2002). Moreover, etazolate exerts anti-

EP

inflammatory effects (Guo et al., 2014; Siopi et al., 2013), probably by acting as an inhibitor of PDE4 (Chasin et al., 1972). It is notheworthy that both PDE4 and GABAA have a potential role

CC

in remyelination after CNS injury (Ghoumari et al., 2003; Gilani et al., 2014; Sun et al., 2012;

A

Syed et al., 2013). All together, these data highlight a suitable model system to study trauma-induced

cerebellar injury and demyelination, and to test active compounds for myelin protection/repair.

19

Acknowledgements This work was supported by fundings from the non-profit organization “Fondation des Gueules Cassées” (grants to GLD and MJT), Paris Descartes University and INSERM (Institut National de la Santé Et de la Recherche Médicale). We acknowledge Patrice Jegouzo and Christophe Tourain for the construction of weight-drop injury device, Jean-Maurice Petit for confocal

IP T

microscopy assistance, Pr Sophie Bernard and Dr Olivier Biondi (INSERM UMR-S 1124, Paris) for their scientific support for the time-lapse microscopy, Dr Anne Simon for her assistance in

Figure editing and Dr Abdel M. Ghoumari (INSERM UMR 788, Paris) for providing the PLP-

SC R

eGFP transgenic mice. The authors acknowledge the assistance of different facilities (animal, molecular biology and imaging facilities) from the Faculty of Basic and Biomedical Sciences

U

(Paris Descartes University).

N

Author Disclosure Statement

A

CC

EP

TE D

M

A

The authors declare no potential competing interests.

20

References Adamchik, Y., Frantseva, M.V., Weisspapir, M., Carlen, P.L., Perez Velazquez, J.L., 2000. Methods to induce primary and secondary traumatic damage in organotypic hippocampal slice cultures. Brain Res Brain Res Protoc 5, 153– 8. Adams, J.H., Graham, D.I., Gennarelli, T.A., 1983. Head injury in man and experimental animals: neuropathology. Acta neurochirurgica. Supplementum 32, 15–30.

IP T

Adembri, C., Bechi, A., Meli, E., Gramigni, E., Venturi, L., Moroni, F., De Gaudio, A.R., Pellegrini-Giampietro, D.E., 2004. Erythropoietin attenuates post-traumatic injury in organotypic hippocampal slices. J. Neurotrauma 21, 1103–1112. https://doi.org/10.1089/0897715041651079 Apps, R., Hawkes, R., 2009. Cerebellar cortical organization: a one-map hypothesis. Nat. Rev. Neurosci. 10, 670– 681. https://doi.org/10.1038/nrn2698

SC R

Armstrong, R.C., Mierzwa, A.J., Marion, C.M., Sullivan, G.M., 2015a. White matter involvement after TBI: Clues to axon and myelin repair capacity. Exp. Neurol. https://doi.org/10.1016/j.expneurol.2015.02.011

Armstrong, R.C., Mierzwa, A.J., Sullivan, G.M., Sanchez, M.A., 2015b. Myelin and Oligodendrocyte Lineage Cells in White Matter Pathology and Plasticity after Traumatic Brain Injury. Neuropharmacology. https://doi.org/10.1016/j.neuropharm.2015.04.029

A

N

U

Bartsch, T., Döhring, J., Reuter, S., Finke, C., Rohr, A., Brauer, H., Deuschl, G., Jansen, O., 2015. Selective neuronal vulnerability of human hippocampal CA1 neurons: lesion evolution, temporal course, and pattern of hippocampal damage in diffusion-weighted MR imaging. J. Cereb. Blood Flow Metab. 35, 1836–1845. https://doi.org/10.1038/jcbfm.2015.137

M

Chasin, M., Harris, D.N., Phillips, M.B., Hess, S.M., 1972. 1-Ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4b)-pyridine-5-carboxylic acid, ethyl ester, hydrochloride (SQ 20009)--a potent new inhibitor of cyclic 3’,5’nucleotide phosphodiesterases. Biochem. Pharmacol. 21, 2443–2450.

TE D

Coburn, M., Maze, M., Franks, N.P., 2008. The neuroprotective effects of xenon and helium in an in vitro model of traumatic brain injury*. Critical Care Medicine 36, 588–595. https://doi.org/10.1097/01.CCM.0B013E3181611F8A6 Croall, I.D., Cowie, C.J.A., He, J., Peel, A., Wood, J., Aribisala, B.S., Mitchell, P., Mendelow, A.D., Smith, F.E., Millar, D., Kelly, T., Blamire, A.M., 2014. White matter correlates of cognitive dysfunction after mild traumatic brain injury. Neurology. https://doi.org/10.1212/WNL.0000000000000666

CC

EP

Cross, D.J., Garwin, G.G., Cline, M.M., Richards, T.L., Yarnykh, V., Mourad, P.D., Ho, R.J.Y., Minoshima, S., 2015. Paclitaxel improves outcome from traumatic brain injury. Brain Res. https://doi.org/10.1016/j.brainres.2015.06.006 Daly, J.W., Hong, O., Padgett, W.L., Shamim, M.T., Jacobson, K.A., Ukena, D., 1988. Non-xanthine heterocycles: activity as antagonists of A1- and A2-adenosine receptors. Biochem. Pharmacol. 37, 655–664.

A

Dent, K.A., Christie, K.J., Bye, N., Basrai, H.S., Turbic, A., Habgood, M., Cate, H.S., Turnley, A.M., 2015. Oligodendrocyte Birth and Death following Traumatic Brain Injury in Adult Mice. PLoS ONE 10, e0121541. https://doi.org/10.1371/journal.pone.0121541 Diaz-Arrastia, R., Kochanek, P.M., Bergold, P., Kenney, K., Marx, C.E., Grimes, C.J.B., Loh, L.T.C.Y., Adam, L.T.C.G.E., Oskvig, D., Curley, K.C., Salzer, W., 2014. Pharmacotherapy of traumatic brain injury: state of the science and the road forward: report of the Department of Defense Neurotrauma Pharmacology Workgroup. J. Neurotrauma 31, 135–158. https://doi.org/10.1089/neu.2013.3019 Doussau, F., Dupont, J.-L., Neel, D., Schneider, A., Poulain, B., Bossu, J.L., 2017. Organotypic cultures of cerebellar slices as a model to investigate demyelinating disorders. Expert Opin Drug Discov 12, 1011–1022.

21

https://doi.org/10.1080/17460441.2017.1356285 Fahlenkamp, A.V., Coburn, M., Czaplik, M., Ryang, Y.-M., Kipp, M., Rossaint, R., Beyer, C., 2011. Expression analysis of the early chemokine response 4 h after in vitro traumatic brain injury. Inflamm. Res. 60, 379– 387. https://doi.org/10.1007/s00011-010-0281-6 Flierl, M.A., Stahel, P.F., Beauchamp, K.M., Morgan, S.J., Smith, W.R., Shohami, E., 2009. Mouse closed head injury model induced by a weight-drop device. Nat Protoc 4, 1328–1337. https://doi.org/10.1038/nprot.2009.148

IP T

Flygt, J., Djupsjö, A., Lenne, F., Marklund, N., 2013. Myelin loss and oligodendrocyte pathology in white matter tracts following traumatic brain injury in the rat. Eur. J. Neurosci. 38, 2153–2165. https://doi.org/10.1111/ejn.12179

SC R

Flygt, J., Ruscher, K., Norberg, A., Mir, A., Gram, H., Clausen, F., Marklund, N., 2018. Neutralization of Interleukin1β following Diffuse Traumatic Brain Injury in the Mouse attenuates the loss of Mature Oligodendrocytes. J. Neurotrauma. https://doi.org/10.1089/neu.2018.5660 Ghoumari, A.M., Ibanez, C., El-Etr, M., Leclerc, P., Eychenne, B., O’Malley, B.W., Baulieu, E.E., Schumacher, M., 2003. Progesterone and its metabolites increase myelin basic protein expression in organotypic slice cultures of rat cerebellum. Journal of Neurochemistry 86, 848–859. https://doi.org/10.1046/j.14714159.2003.01881.x

A

N

U

Gilani, A.A., Dash, R.P., Jivrajani, M.N., Thakur, S.K., Nivsarkar, M., 2014. Evaluation of GABAergic Transmission Modulation as a Novel Functional Target for Management of Multiple Sclerosis: Exploring Inhibitory Effect of GABA on Glutamate-Mediated Excitotoxicity. Adv Pharmacol Sci 2014, 632376. https://doi.org/10.1155/2014/632376

M

Guo, J., Lin, P., Zhao, X., Zhang, J., Wei, X., Wang, Q., Wang, C., 2014. Etazolate abrogates the lipopolysaccharide (LPS)-induced downregulation of the cAMP/pCREB/BDNF signaling, neuroinflammatory response and depressive-like behavior in mice. Neuroscience 263, 1–14. https://doi.org/10.1016/j.neuroscience.2014.01.008

TE D

Herrera, J.J., Bockhorst, K.H., Kondraganti, S., Stertz, L., DeQuevedo, J.L., Narayana, P.A., 2016. Acute white matter tract damage following frontal mild traumatic brain injury. J. Neurotrauma. https://doi.org/10.1089/neu.2016.4407

EP

Johnson, V.E., Stewart, J.E., Begbie, F.D., Trojanowski, J.Q., Smith, D.H., Stewart, W., 2013. Inflammation and white matter degeneration persist for years after a single traumatic brain injury. Brain 136, 28–42. https://doi.org/10.1093/brain/aws322

CC

Kinnunen, K.M., Greenwood, R., Powell, J.H., Leech, R., Hawkins, P.C., Bonnelle, V., Patel, M.C., Counsell, S.J., Sharp, D.J., 2011. White matter damage and cognitive impairment after traumatic brain injury. Brain 134, 449–463. https://doi.org/10.1093/brain/awq347 Kondiles, B.R., Horner, P.J., 2018. Myelin plasticity, neural activity, and traumatic neural injury. Dev Neurobiol 78, 108–122. https://doi.org/10.1002/dneu.22540

A

Krassioukov, A.V., Ackery, A., Schwartz, G., Adamchik, Y., Liu, Y., Fehlings, M.G., 2002. An in vitro model of neurotrauma in organotypic spinal cord cultures from adult mice. Brain Res. Brain Res. Protoc. 10, 60–68. Kumaria, A., Tolias, C.M., 2008. In vitro models of neurotrauma. Br J Neurosurg 22, 200–206. https://doi.org/10.1080/02688690701772413 Llufriu-Dabén, G., Carrete, A., Chierto, E., Mailleux, J., Camand, E., Simon, A., Vanmierlo, T., Rose, C., Allinquant, B., Hendriks, J.J.A., Massaad, C., Meffre, D., Jafarian-Tehrani, M., 2018. Targeting demyelination via αsecretases promoting sAPPα release to enhance remyelination in central nervous system. Neurobiol. Dis. 109, 11–24. https://doi.org/10.1016/j.nbd.2017.09.008

22

Lotocki, G., de Rivero Vaccari, J.P., de Rivero Vaccari, J., Alonso, O., Molano, J.S., Nixon, R., Safavi, P., Dietrich, W.D., Bramlett, H.M., 2011. Oligodendrocyte vulnerability following traumatic brain injury in rats. Neurosci. Lett. 499, 143–148. https://doi.org/10.1016/j.neulet.2011.05.056 Maas, A.I.R., Menon, D.K., Adelson, P.D., Andelic, N., Bell, M.J., et al., 2017. Traumatic brain injury: integrated approaches to improve prevention, clinical care, and research. Lancet Neurol 16, 987–1048. https://doi.org/10.1016/S1474-4422(17)30371-X Majdan, M., Plancikova, D., Brazinova, A., Rusnak, M., Nieboer, D., Feigin, V., Maas, A., 2016. Epidemiology of traumatic brain injuries in Europe: a cross-sectional analysis. Lancet Public Health 1, e76–e83. https://doi.org/10.1016/S2468-2667(16)30017-2

IP T

Mallon, B.S., Shick, H.E., Kidd, G.J., Macklin, W.B., 2002. Proteolipid promoter activity distinguishes two populations of NG2-positive cells throughout neonatal cortical development. J Neurosci 22, 876–85.

SC R

Marcade, M., Bourdin, J., Loiseau, N., Peillon, H., Rayer, A., Drouin, D., Schweighoffer, F., Désiré, L., 2008. Etazolate, a neuroprotective drug linking GABA(A) receptor pharmacology to amyloid precursor protein processing. J. Neurochem. 106, 392–404. https://doi.org/10.1111/j.1471-4159.2008.05396.x Miller, A.P., Shah, A.S., Aperi, B.V., Budde, M.D., Pintar, F.A., Tarima, S., Kurpad, S.N., Stemper, B.D., GlavaskiJoksimovic, A., 2015. Effects of blast overpressure on neurons and glial cells in rat organotypic hippocampal slice cultures. Front Neurol 6, 20. https://doi.org/10.3389/fneur.2015.00020

N

U

Miller, A.P., Shah, A.S., Aperi, B.V., Kurpad, S.N., Stemper, B.D., Glavaski-Joksimovic, A., 2017. Acute death of astrocytes in blast-exposed rat organotypic hippocampal slice cultures. PLoS ONE 12, e0173167. https://doi.org/10.1371/journal.pone.0173167

A

Morganti-Kossmann, M.C., Satgunaseelan, L., Bye, N., Kossmann, T., 2007. Modulation of immune response by head injury. Injury 38, 1392–400. https://doi.org/10.1016/j.injury.2007.10.005

M

Morrison, B., Elkin, B.S., Dollé, J.-P., Yarmush, M.L., 2011. In vitro models of traumatic brain injury. Annu Rev Biomed Eng 13, 91–126. https://doi.org/10.1146/annurev-bioeng-071910-124706

TE D

Nalivaeva, N.N., Turner, A.J., 2013. The amyloid precursor protein: a biochemical enigma in brain development, function and disease. FEBS Lett. 587, 2046–2054. https://doi.org/10.1016/j.febslet.2013.05.010 Notterpek, L.M., Bullock, P.N., Malek-Hedayat, S., Fisher, R., Rome, L.H., 1993. Myelination in cerebellar slice cultures: development of a system amenable to biochemical analysis. J. Neurosci. Res. 36, 621–634. https://doi.org/10.1002/jnr.490360603

EP

Park, E., Ai, J., Baker, A.J., 2007. Cerebellar injury: clinical relevance and potential in traumatic brain injury research. Prog. Brain Res. 161, 327–338. https://doi.org/10.1016/S0079-6123(06)61023-6

CC

Potts, M.B., Adwanikar, H., Noble-Haeusslein, L.J., 2009. Models of traumatic cerebellar injury. Cerebellum 8, 211–221. https://doi.org/10.1007/s12311-009-0114-8 Raghupathi, R., 2004. Cell death mechanisms following traumatic brain injury. Brain Pathol. 14, 215–222.

A

Reeber, S.L., Otis, T.S., Sillitoe, R.V., 2013. New roles for the cerebellum in health and disease. Front Syst Neurosci 7, 83. https://doi.org/10.3389/fnsys.2013.00083 Rieger, A.M., Nelson, K.L., Konowalchuk, J.D., Barreda, D.R., 2011. Modified annexin V/propidium iodide apoptosis assay for accurate assessment of cell death. J Vis Exp. https://doi.org/10.3791/2597 Rodriguez-Grande, B., Obenaus, A., Ichkova, A., Aussudre, J., Bessy, T., Barse, E., Hiba, B., Catheline, G., Barrière, G., Badaut, J., 2018. Gliovascular changes precede white matter damage and long-term disorders in juvenile mild closed head injury. Glia. https://doi.org/10.1002/glia.23336 Saatman, K.E., Duhaime, A.C., Bullock, R., Maas, A.I., Valadka, A., Manley, G.T., Workshop Scientific, T., Advisory

23

Panel, M., 2008. Classification of traumatic brain injury for targeted therapies. Journal of neurotrauma 25, 719–38. https://doi.org/10.1089/neu.2008.0586 Scheller, A., Bai, X., Kirchhoff, F., 2017. The Role of the Oligodendrocyte Lineage in Acute Brain Trauma. Neurochem. Res. https://doi.org/10.1007/s11064-017-2343-4 Schmidt, O.I., Heyde, C.E., Ertel, W., Stahel, P.F., 2005. Closed head injury--an inflammatory disease? Brain research. Brain research reviews 48, 388–99. https://doi.org/10.1016/j.brainresrev.2004.12.028 Shaw, K., MacKinnon, M.A., Raghupathi, R., Saatman, K.E., Mclntosh, T.K., Graham, D.I., 2001. TUNEL-positive staining in white and grey matter after fatal head injury in man. Clin. Neuropathol. 20, 106–112.

IP T

Shi, H., Hu, X., Leak, R.K., Shi, Y., An, C., Suenaga, J., Chen, J., Gao, Y., 2015. Demyelination as a Rational Therapeutic Target for Ischemic or Traumatic Brain Injury. Exp. Neurol. https://doi.org/10.1016/j.expneurol.2015.03.017

SC R

Singleton, R.H., Stone, J.R., Okonkwo, D.O., Pellicane, A.J., Povlishock, J.T., 2001. The immunophilin ligand FK506 attenuates axonal injury in an impact-acceleration model of traumatic brain injury. J. Neurotrauma 18, 607–614. https://doi.org/10.1089/089771501750291846

U

Siopi, E., Llufriu-Dabén, G., Cho, A.H., Vidal-Lletjós, S., Plotkine, M., Marchand-Leroux, C., Jafarian-Tehrani, M., 2013. Etazolate, an α-secretase activator, reduces neuroinflammation and offers persistent neuroprotection following traumatic brain injury in mice. Neuropharmacology 67, 183–192. https://doi.org/10.1016/j.neuropharm.2012.11.009

A

N

Spanos, G.K., Wilde, E.A., Bigler, E.D., Cleavinger, H.B., Fearing, M.A., Levin, H.S., Li, X., Hunter, J.V., 2007. cerebellar atrophy after moderate-to-severe pediatric traumatic brain injury. AJNR Am J Neuroradiol 28, 537–542.

M

Staal, J.A., Vickers, J.C., 2011. Selective vulnerability of non-myelinated axons to stretch injury in an in vitro coculture system. J. Neurotrauma 28, 841–847. https://doi.org/10.1089/neu.2010.1658

TE D

Staffa, K., Ondruschka, B., Franke, H., Dreßler, J., 2012. Cerebellar Gene Expression following Human Traumatic Brain Injury. Journal of Neurotrauma 29, 2716–2721. https://doi.org/10.1089/neu.2011.2246 Sun, X., Liu, Y., Liu, B., Xiao, Z., Zhang, L., 2012. Rolipram promotes remyelination possibly via MEK-ERK signal pathway in cuprizone-induced demyelination mouse. Exp. Neurol. 237, 304–311. https://doi.org/10.1016/j.expneurol.2012.07.011

EP

Sundstrom, L., Morrison, B., Bradley, M., Pringle, A., 2005. Organotypic cultures as tools for functional screening in the CNS. Drug Discov. Today 10, 993–1000. https://doi.org/10.1016/S1359-6446(05)03502-6

CC

Syed, Y.A., Baer, A., Hofer, M.P., González, G.A., Rundle, J., Myrta, S., Huang, J.K., Zhao, C., Rossner, M.J., Trotter, M.W.B., Lubec, G., Franklin, R.J.M., Kotter, M.R., 2013. Inhibition of phosphodiesterase-4 promotes oligodendrocyte precursor cell differentiation and enhances CNS remyelination. EMBO Mol Med 5, 1918– 1934. https://doi.org/10.1002/emmm.201303123

A

Taib, T., Leconte, C., Van Steenwinckel, J., Cho, A.H., Palmier, B., Torsello, E., Lai Kuen, R., Onyeomah, S., Ecomard, K., Benedetto, C., Coqueran, B., Novak, A.-C., Deou, E., Plotkine, M., Gressens, P., Marchand-Leroux, C., Besson, V.C., 2017. Neuroinflammation, myelin and behavior: Temporal patterns following mild traumatic brain injury in mice. PLoS ONE 12, e0184811. https://doi.org/10.1371/journal.pone.0184811 Thompson, S.-A., Wingrove, P.B., Connelly, L., Whiting, P.J., Wafford, K.A., 2002. Tracazolate reveals a novel type of allosteric interaction with recombinant gamma-aminobutyric acid(A) receptors. Mol. Pharmacol. 61, 861–869. Wang, G., Shi, Y., Jiang, X., Leak, R.K., Hu, X., Wu, Y., Pu, H., Li, W.-W., Tang, B., Wang, Y., Gao, Y., Zheng, P., Bennett, M.V.L., Chen, J., 2015. HDAC inhibition prevents white matter injury by modulating microglia/macrophage polarization through the GSK3β/PTEN/Akt axis. Proc. Natl. Acad. Sci. U.S.A.

24

https://doi.org/10.1073/pnas.1501441112 Wang, X., Michaelis, E.K., 2010. Selective neuronal vulnerability to oxidative stress in the brain. Front Aging Neurosci 2, 12. https://doi.org/10.3389/fnagi.2010.00012 Wang, Z., Wu, W., Liu, Y., Wang, T., Chen, X., Zhang, J., Zhou, G., Chen, R., 2016. Altered Cerebellar White Matter Integrity in Patients with Mild Traumatic Brain Injury in the Acute Stage. PLoS ONE 11, e0151489. https://doi.org/10.1371/journal.pone.0151489

A

CC

EP

TE D

M

A

N

U

SC R

IP T

Weber, A.M., Torres, C., Rauscher, A., 2018. Imaging the Role of Myelin in Concussion. Neuroimaging Clin. N. Am. 28, 83–90. https://doi.org/10.1016/j.nic.2017.09.005

25

EP

CC

A TE D

IP T

SC R

U

N

A

M

Figr-1

26

EP

CC

A TE D

IP T

SC R

U

N

A

M

Figr-2

27

EP

CC

A TE D

IP T

SC R

U

N

A

M

Figr-3

28

EP

CC

A TE D

IP T

SC R

U

N

A

M

Figr-4

29

EP

CC

A TE D

IP T

SC R

U

N

A

M

Figr-5

30

Figr-6 Figure Legends Fig. 1. Weight-drop device to induce traumatic injury. (A) The tissue injury device was designed and adapted to induce traumatic injury in organotypic cerebellar slices. Injury was provoked when the stylus (324 mg, 1 mm of diameter) impacted the cerebellar slice from a 5

IP T

mm, 7 mm or 10 mm height. The stylus was round and smooth to avoid tissue damage or perforation. (B) A magnification of the stylus and the organotypic cerebellar slices pointed by

SC R

arrow and arrowhead, respectively.

Fig. 2. Experimental timeline. Organotypic cerebellar slices were obtained from C57BL/6 or transgenic PLP-eGFP mouse pups (P8-P10). Weight-drop traumatic injury was performed

U

from a 7 mm height at 7 DIV. Few minutes prior to the traumatic insult, slices were treated with

N

experimental serum-free medium containing PI to assess tissue lesion, and etazolate (2 µM)

A

or its vehicle (PBS) to assess the effect of compound against tissue injury. PI-fluorescence

M

was measured at 6h, 24h and 72h post-injury. GFP-fluorescence was measured 1h before the insult (-1h), and at 6h, 24h and 72h after injury along with PI-fluorescence by live imaging. The

TE D

slices were then fixed and processed for immunohistochemistry, or collected to study myelin proteins by Western blotting. DIV: Days in vitro; PI: propidium iodide; GFP: green fluorescent protein.

EP

Fig. 3. Time course of trauma-induced injury in organotypic cerebellar slices: protective

CC

effect of etazolate. (A) Weight-drop traumatic injury was performed from a 7 mm height at 7 DIV. Representative images of cerebellar slices were highlighted by PI uptake, delimitated by

A

dotted lines. Vehicle- or etazolate-treated control slices did not show an increase of PIfluorescence within 72h of treatment. PI-fluorescence dramatically increased at the site of the injury of vehicle-treated injured slices at 6h, 24h and 72h post-injury. Etazolate attenuated PI uptake at all experimental time points studied. Scale bar: 500 µm. (B) Quantification of cerebellar injury was evaluated at 6h, 24h and 72h for the same slice by measuring the PIfluorescence. The traumatic insult significantly increased cerebellar injury at 6h, progressing

31

until 72h post-injury. Post-traumatic injury was significantly attenuated at all time points in the presence of etazolate. Results are expressed as mean  s.e.m (n=28-40 slices per condition obtained from seven independent experiments). aP=0.054; **P<0.01; ****P<0.0001. Ctl: control slices; Vehicle: PBS; Etaz: etazolate 2 µM; PI: propidium iodide. Fig. 4. Qualitative analysis of ex vivo trauma on Purkinje cells, myelin sheaths and

IP T

myelin proteins. (A) Representative confocal images of vehicle-treated and etazolate-treated control slices showed intact Purkinje cells (Calbindin, in blue) and myelin sheaths (MAG, in

SC R

green). At 72h post-injury, vehicle-treated injured slices showed loss of Purkinje cells bodies and axons, as well as myelin sheath loss in the injured area (open arrowhead), whereas etazolate-treated injured slices showed an enhanced staining of both markers (full

U

arrowheads). Scale bar: 50 µm. (B) Representative immunoblot showing a decrease of the

N

amounts of specific myelin protein MBP within the injured area from vehicle-treated injured

A

slices at 72h. Treatment with etazolate counteracts trauma-induced MBP protein loss. (C)

M

Quantitative analysis of relative amount of total MBP protein (18 and 21 kDa) versus control group. Ctl: control slices; Veh: vehicle (PBS); Etaz: etazolate 2 µM; MAG: myelin associated

TE D

glycoprotein. The amount of 30 μg of proteins was loaded for each condition. Proteins were extracted from 5-8 slices (from 2 cerebella) and three independent experiments were performed.

EP

Fig. 5. Time course of trauma-induced demyelination in organotypic cerebellar slices:

CC

protective effect of etazolate. Weight-drop traumatic injury was performed from a 7 mm height at 7 DIV. (A) Representative images of the PLP-eGFP slices, delimitated by dotted

A

lines, imaged at 1h before injury (-1h) and at 72h post-injury (72h), scale bar: 500 µm. A 10X magnification of the quantification area is presented in column 2 and 4, scale bar: 50 µm. (B) Quantitative analysis showed that vehicle- or etazolate-treated control slices did not show a modification of GFP-fluorescence within 72h of treatment. GFP-fluorescence dropped at the site of the injury of vehicle-treated injured slices at 6h, 24h and 72h post-injury. Etazolate treatment was able to counteract the GFP-fluorescence loss on injured slices at all time points

32

studied. Data are expressed as the percentage of GFP-fluorescence for each slice at each time point, reported to the GFP-fluorescence of the same slice at 1h before injury (-1h). Results are expressed as mean  s.e.m (n=11-19 slices per condition obtained from three independent experiments). aP=0.054; bP=0.053; *P<0.05; ****P<0.0001. Ctl: control slices; Vehicle: PBS; Etaz: etazolate 2 µM; PLP: proteolipid protein; GFP: green fluorescent protein; PI: propidium

IP T

iodide. Table 1. Time course of PI uptake in cerebellar slices following traumatic injury from

SC R

different heights using a weight-drop device. Traumatic injury was induced by the drop of

a stylus of 324 mg (1 mm of diameter) from a 5, 7 or 10 mm height. Tissue lesion was quantified as an increase of PI-fluorescence at 6h, 24h and 72h post-injury. Results are expressed as

A

CC

EP

TE D

M

A

N

*P<0.05; **P<0.01; ***P<0.001 versus control slices.

U

mean  s.e.m (n=3-9 slices per condition obtained from two independent experiments).

33

Time after injury Height

6h

24h

72h

Control

1779 ± 1406

3648 ± 460

4072 ± 1631

1.78 x106 ± 0.8 x106 **

1.85 x106 ± 0.8 x106 **

**

1.33 x106 ± 0.6 x106

7 mm

1.28 x106 ± 0.2 x106 ***

2.30 x106 ± 0.2 x106 ***

2.28 x106 ± 0.05 x106 *

10 mm

1.62 x106 ± 0.3 x106

***

2.79 x106 ± 0.5 x106 ***

2.25 x106 ± 0.5 x106 ***

A

CC

EP

TE D

M

A

N

U

SC R

IP T

5 mm

34