Elsevier

Experimental Neurology

Volume 306, August 2018, Pages 250-259
Experimental Neurology

Research Paper
Systemic epothilone D improves hindlimb function after spinal cord contusion injury in rats

https://doi.org/10.1016/j.expneurol.2018.01.018Get rights and content

Highlights

  • Systemic Epo D injections are beneficial after moderate spinal cord injury.

  • Epo D does not enhance serotonergic axon growth below the lesion.

  • Epo D has no neuroprotective effects.

Abstract

Following a spinal cord injury (SCI) a growth aversive environment forms, consisting of a fibroglial scar and inhibitory factors, further restricting the already low intrinsic growth potential of injured adult central nervous system (CNS) neurons. Previous studies have shown that local administration of the microtubule-stabilizing drug paclitaxel or epothilone B (Epo B) reduce fibrotic scar formation and axonal dieback as well as induce axonal growth/sprouting after SCI. Likewise, systemic administration of Epo B promoted functional recovery. In this study, we investigated the effects of epothilone D (Epo D), an analog of Epo B with a possible greater therapeutic index, on fibrotic scarring, axonal sprouting and functional recovery after SCI. Delayed systemic administration of Epo D after a moderate contusion injury (150 kDyn) in female Fischer 344 rats resulted in a reduced number of footfalls when crossing a horizontal ladder at 4 and 8 weeks post-injury. Hindlimb motor function assessed with the BBB open field locomotor rating scale and Catwalk gait analysis were not significantly altered. Moreover, formation of laminin positive fibrotic scar tissue and 5-HT positive serotonergic fiber length caudal to the lesion site were not altered after treatment with Epo D. These findings recapitulate a functional benefit after systemic administration of a microtubule-stabilizing drug in rat contusion SCI.

Introduction

Traumatic spinal cord injury (SCI) results in sensorimotor and autonomic deficits due to the disruption of descending motor and ascending sensory pathways. After the initial insult, secondary degeneration involving various pathophysiological mechanisms leads to the formation of fluid filled cavities, loss of gray and white matter and the formation of a glial and fibrotic scar. Furthermore, retrograde degeneration including dieback of axons, formation of retraction bulbs and disorganization of microtubule exacerbate the primary injury (Erturk et al., 2007).

Stabilizing microtubules inhibits proliferation of cells, which has been exploited in drug development for cancer therapies. The stabilization of microtubules in neurons is vital for axonal transport and function (Brunden et al., 2010; Garcia and Cleveland, 2001; Goedert and Jakes, 2005). Therefore microtubule stabilizing drugs have been investigated in animal models of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and schizophrenia to abate degenerating axons and promote regeneration (Andrieux et al., 2006; Ballatore et al., 2012; Brunden et al., 2010; Cartelli et al., 2013; Daoust et al., 2014; Fournet et al., 2012).

The effect of the microtubule stabilizing anti-cancer drug paclitaxel on microtubule dynamics is concentration-dependent. High concentrations, which are typically used for cancer treatment, over-stabilize microtubules, thus blocking microtubule dynamics, which in turn suppresses axon elongation (Erturk et al., 2007; Sengottuvel and Fischer, 2011; Sengottuvel et al., 2011). In contrast, low concentrations of paclitaxel lead to moderate microtubule stabilization in axonal growth cones, which still allows microtubule polymerization at the plus end (Derry et al., 1995; Derry et al., 1997; Witte et al., 2008), enhances axonal growth in cultured neurons (Hellal et al., 2011; Sengottuvel et al., 2011) and preserves microtubule stability and bundling in injured spinal cord axons (Erturk et al., 2007). In rodent SCI and optic nerve injury models, paclitaxel inhibits fibrotic scarring and deposition of inhibitory CSPGs (Chondroitin sulfate proteoglycans) in the lesion site and promotes axonal regeneration as well as functional recovery (Erturk et al., 2007; Hellal et al., 2011; Perez-Espejo et al., 1996; Popovich et al., 2014; Sengottuvel et al., 2011). Moreover, low paclitaxel concentrations have been shown to at least transiently delay macrophage infiltration and astrocyte proliferation around the lesion site after optic nerve injury (Sengottuvel and Fischer, 2011; Sengottuvel et al., 2011).

In contrast to paclitaxel, which cannot cross the blood-brain barrier (BBB), some epothilones are BBB permeable while possessing comparable biological effects and sharing a common binding site (Giannakakou et al., 2000). Thus they are more suitable candidates for treatment of neurological disorders and injuries. Epothilones already have U.S. Food and Drug Administration (FDA) approval for cancer treatment (Brogdon et al., 2014; Cheng et al., 2008; Goodin et al., 2004).

Both analogs, epothilone B (Epo B) and epothilone (Epo D), act in a dose dependent manner on microtubule stabilization similar to paclitaxel (Altmann et al., 2000a; Altmann et al., 2000b; Ballatore et al., 2012; Cheng et al., 2008; Goodin et al., 2004; Muhlradt and Sasse, 1997). A recent study using an in vitro axotomy model showed that the exposure of neurons to Epo D increased axonal sprouting without affecting their viability or metabolic function (Brizuela et al., 2015). The application of Epo D in animal models of tauopathies such as Alzheimer's disease improved axonal transport, decreased tau neuropathology, reduced neuronal loss and axonal dystrophy paralleled by amelioration of cognitive deficits (Barten et al., 2012; Brill et al., 2016; Brunden et al., 2010; Lou et al., 2014; Zhang et al., 2012). The systemic administration of Epo B in a moderate rat spinal cord contusion injury reduced fibrotic scar formation by interfering with fibroblast migration to the lesion site, promoted axonal growth by stabilizing microtubules and reduced the number of footfalls when crossing a regular spaced horizontal ladder (Ruschel et al., 2015).

Given that clinical trials Epo D reported a better safety profile than Epo B including a greater therapeutic index (Chou et al., 1998; Fumoleau et al., 2007; Goodin et al., 2004) we investigated potential effects of Epo D in a rat contusion SCI model. We observed that systemic administration of Epo D results in improved skilled hindlimb function after a moderate spinal cord contusion injury.

Section snippets

Animal subjects

Adult female Fischer 344 rats (Charles River Deutschland GmbH, Sulzfeld, Germany, Envigo, Cambridgeshire, UK; Janvier Labs, Saint-Berthevin Cedex, France) weighing 160–180 g were used for all in vivo experiments. Experiments were carried out in accordance with the European Union Directive (2010/63/EU) and institutional guidelines. Animals had ad libitum access to food and water throughout the study.

Surgical procedures and treatment

For all surgical procedures, animals were anesthetized using a cocktail of ketamine (62.5 mg/kg;

Differential tissue displacement generates distinct injury severity cohorts

Following a 150 kDyn T9 contusion injury in adult female Fischer 344 rats, displacement curves were examined for consistency. The Infinite Horizon (IH) impactor is controlled by force usually resulting in consistent displacement of a given directed force, although some variation can occur. Because both force and displacement lead to anatomical changes and as a consequence affect functional outcomes after contusive SCI (Ghasemlou et al., 2005), it is important to examine tissue displacement in

Discussion

In addition to the disruption of ascending and descending pathways, secondary injury mechanisms involving Wallerian degeneration and the formation of a fibroglial scar contribute to the functional deficits after SCI. Previous studies indicate that the administration of the microtubule-stabilizing drugs (paclitaxel and Epo B) result in reduced fibrotic scar formation, less axonal dieback and improvements in functional recovery after moderate SCI (Hellal et al., 2011; Ruschel et al., 2015).

The

Conclusions

Overall, the present study revealed functional improvement after Epo D administration, comparable to the benefit observed in previous studies investigating the microtubule stabilizing drugs Epo B and paclitaxel (Hellal et al., 2011; Perez-Espejo et al., 1996; Ruschel et al., 2015). However, the underlying structural correlates could not be clearly identified. Therefore, future experiments will expand the focus to relevant descending and ascending axonal pathways other than serotoninergic axons

Acknowledgments

This work was supported by the Olympia-Morata-Program at Heidelberg University to BS and the Interdisciplinary Neurobehavioral Core (INBC) (AZ42-04HV.MED(15)/6/1) in Heidelberg.

Author contributions

F.B. initiated the continuation study and shared the Epo D results of his lab prior to initiation of this study. F.B. and J.R. helped to design the experiments. J.R. advised the Heidelberg laboratory on drug dosing, administration and on the contusion injury procedure. F.B. and J.R. were not present during the experiments and did not participate in the data analysis. B.S. and A.B. performed all surgeries and administered the drug. B.S. performed the behavioral testing and analysis, perfusions,

Author disclosure statement

H. Witte, A. Ertürk, F. Hellal, and F.B. filed a patent on the use of microtubule-stabilizing compounds for the treatment of lesions of CNS axons (European Patent no. 1858498; European patent application EP 11 00 9155.0; U.S. patent application 11/908,118). The authors declare no competing financial interests.

References (61)

  • P.G. Popovich et al.

    Independent evaluation of the effects of glibenclamide on reducing progressive hemorrhagic necrosis after cervical spinal cord injury

    Exp. Neurol.

    (2012)
  • P.G. Popovich et al.

    A reassessment of a classic neuroprotective combination therapy for spinal cord injured rats: LPS/pregnenolone/indomethacin

    Exp. Neurol.

    (2012)
  • P.G. Popovich et al.

    Independent evaluation of the anatomical and behavioral effects of Taxol in rat models of spinal cord injury

    Exp. Neurol.

    (2014)
  • P. Schucht et al.

    Anatomical correlates of locomotor recovery following dorsal and ventral lesions of the rat spinal cord

    Exp. Neurol.

    (2002)
  • A.A. Webb et al.

    Fischer (F-344) rats have different morphology, sensorimotor and locomotor abilities compared to Lewis, Long-Evans, Sprague-Dawley and Wistar rats

    Behav. Brain Res.

    (2003)
  • K.H. Altmann et al.

    Epothilones and related structures—a new class of microtubule inhibitors with potent in vivo antitumor activity

    Biochim. Biophys. Acta

    (2000)
  • C. Ballatore et al.

    Microtubule stabilizing agents as potential treatment for Alzheimer's disease and related neurodegenerative tauopathies

    J. Med. Chem.

    (2012)
  • F.M. Bareyre et al.

    The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats

    Nat. Neurosci.

    (2004)
  • D.M. Barten et al.

    Hyperdynamic microtubules, cognitive deficits, and pathology are improved in tau transgenic mice with low doses of the microtubule-stabilizing agent BMS-241027

    J. Neurosci.

    (2012)
  • D.M. Basso et al.

    A sensitive and reliable locomotor rating scale for open field testing in rats

    J. Neurotrauma

    (1995)
  • T.M. Beer et al.

    Phase II study of KOS-862 in patients with metastatic androgen independent prostate cancer previously treated with docetaxel

    Investig. New Drugs

    (2007)
  • R. van den Brand et al.

    Restoring voluntary control of locomotion after paralyzing spinal cord injury

    Science

    (2012)
  • C.F. Brogdon et al.

    Development of other microtubule-stabilizer families: the epothilones and their derivatives

    Anti-Cancer Drugs

    (2014)
  • K.R. Brunden et al.

    Epothilone D improves microtubule density, axonal integrity, and cognition in a transgenic mouse model of tauopathy

    J. Neurosci.

    (2010)
  • E. Brustein et al.

    Recovery of locomotion after ventral and ventrolateral spinal lesions in the cat. II. Effects of noradrenergic and serotoninergic drugs

    J. Neurophysiol.

    (1999)
  • D. Cartelli et al.

    Microtubule alterations occur early in experimental parkinsonism and the microtubule stabilizer epothilone D is neuroprotective

    Sci. Rep.

    (2013)
  • K.L. Cheng et al.

    Novel microtubule-targeting agents - the epothilones

    Biologics

    (2008)
  • T.C. Chou et al.

    Desoxyepothilone B: an efficacious microtubule-targeted antitumor agent with a promising in vivo profile relative to epothilone B

    Proc. Natl. Acad. Sci. U. S. A.

    (1998)
  • G. Courtine et al.

    Recovery of supraspinal control of stepping via indirect propriospinal relay connections after spinal cord injury

    Nat. Med.

    (2008)
  • W.B. Derry et al.

    Substoichiometric binding of taxol suppresses microtubule dynamics

    Biochemistry

    (1995)
  • Cited by (43)

    • Recognition of necrotic regions in MRI images of chronic spinal cord injury based on superpixel

      2023, Computer Methods and Programs in Biomedicine
      Citation Excerpt :

      Although there is no final conclusion on the effect of glial scar in the process of nerve regeneration, one of the key point to repair the tissue after chronic SCI is to remove the obstacles of regeneration, which means that it is necessary to eliminate the influence of glial scar or cystic cavity before treatment [15]. At present, commonly methods to inhibit glial scar in experimental studies include enzyme digestion [16], antibody blocking [17], or homologous cell transplantation [18]. The feasibility of applying these methods in clinical treatment remains to be confirmed.

    • RhoA drives actin compaction to restrict axon regeneration and astrocyte reactivity after CNS injury

      2021, Neuron
      Citation Excerpt :

      Therefore, ablation of RhoA in neurons allows axon regeneration through a defined cellular cascade that ultimately enables microtubule protrusion in the axon tip, propelling it forward (Dupraz et al., 2019; Santos et al., 2020). Thus, RhoA interconnects extracellular inhibitory signals to microtubules, whose pharmacological stabilization through Taxol and epothilones has beneficial effects after CNS injury (Ertürk et al., 2007; Hellal et al., 2011; Kondo et al., 2019; Kugler et al., 2020; Nagai et al., 2016; Ruschel and Bradke, 2018; Ruschel et al., 2015; Sandner et al., 2018; Zhao et al., 2017). Even though RhoA is a key mediator of axon growth inhibition, additional intracellular mediators of outgrowth inhibition are involved.

    View all citing articles on Scopus
    View full text