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Neuroprotective effect of acute prior inflammation with lipopolysaccharide for adult male rat facial motoneurones
Accepted Manuscript Research report Neuroprotective effect of acute prior inflammation with lipopolysaccharide for adult male rat facial motoneurones Viythia Katharesan, Shane Deery, Ian P Johnson PII: DOI: Reference:
S0006-8993(18)30309-3 https://doi.org/10.1016/j.brainres.2018.05.039 BRES 45822
To appear in:
Brain Research
Received Date: Revised Date: Accepted Date:
3 January 2018 22 May 2018 25 May 2018
Please cite this article as: V. Katharesan, S. Deery, I.P. Johnson, Neuroprotective effect of acute prior inflammation with lipopolysaccharide for adult male rat facial motoneurones, Brain Research (2018), doi: https://doi.org/10.1016/ j.brainres.2018.05.039
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Viythia Katharesan, Shane Deery, and Ian P Johnson* Discipline of Anatomy and Pathology The University of Adelaide Australia SA5005 Neuroprotective effect of acute prior inflammation with lipopolysaccharide for adult male rat facial motoneurones
*Corresponding author: Email: [email protected] Telephone: +61 8 8313 4526
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ABBREVIATIONS CLSM: Confocal laser scanning microscope GM-CSF: Granulocyte Macrophage Colony Stimulating Factor LPS: Lipopolysaccharide IFN-γ: Interferon γ IL: Interleukin TNF-α: Tumour Necrosis Factor α MND: Motor Neuron Disease CSF: Cerebrospinal fluid
ABSTRACT Increases in inflammatory cytokines are reported to have both neuroprotective and neurotoxic effects depending on the type and age of neurones studied. This study aimed to determine the effect of experimental inflammation induced by Lipopolysaccharide (LPS) on the survival of injured male adult rat facial motoneurones. Time- and dose- response studies were done to optimise the LPS administration time and dose, to best correlate with inflammatory levels previously reported for aged rats. 12 cytokines were assayed through multiplex analysis. 24 hours after intraperitoneal injection of 0.5mg/kg Lipopolysaccharide in rats, IL-1β, IL-5 and IL12p70 levels were elevated, with no observed LPS-associated sickness behaviour. In other groups of 5-6 adult rats, the facial nerve was either crushed (as mild injury) or avulsed (as severe
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injury) after the LPS priming injection. Stereology revealed that most motoneurones survived 28 days after nerve crush only and LPS- or saline-priming preceding nerve crush. Most motoneurones died following nerve avulsion only, whereas over half survived when LPSpriming preceded nerve avulsion. We suggest that elevated levels of experimental inflammation are neuroprotective for severely injured adult male rat facial motoneurones.
KEY WORDS Neuroinflammation, Cytokines, Stereology, Motor neuron, Rescue, Facial Nerve
1 INTRODUCTION
Inflammatory status has a major impact on neuronal survival, but whether it promotes or prevents neuronal degeneration is unclear. Elevated inflammatory levels due to challenge by pathogens or by trauma have been associated with neuronal dysfunction and degeneration in both developing- and adult- animals [1-3]. Increased inflammation with ageing animals has been implicated in the pathogenesis of age-related neurodegenerative disease [4, 5]. In contrast, increased inflammatory levels after LPS priming (intraperitoneally) have been found to provide neuroprotection in experimental models of stroke [6] and traumatic brain injury [7-9]. Furthermore, local expression of IL-10 by astrocytes increases the survival of axotomised/injured motoneurones [10]. In age-related neurodegenerative diseases, there is increasing evidence to show that neuroinflammation may be resultant, rather than causative, of disease [11], and it has been proposed that microglial activation with neuroinflammaging may in fact be neuroprotective [4, 12]. Part of this confusion about the role of inflammation in neurodegeneration and neuronal 3
survival may be due to the difficulty of comparing studies using varying models of inflammation and the inherent limitation of studying neuronal death in ageing animals, which are very difficult to obtain and maintain. Sustaining experimental geriatric animals for long periods, while keeping in line with animal ethics, is difficult [13] and unlike their young counterparts, ageing animals’ motoneurones can be more robust and resistant to experimental injury [14]. Thus, there tends to be a preference to work on young animals, which are easier to maintain and in which, it is easier to provoke rapid and substantial neuronal death. This inevitably lends young animals as a productive, but unsuited model that leaves extrapolation of data non-transferable to neurodegenerative conditions in which the ageing demographic, and therefore ageing neurones are affected.
Lipopolysaccharide is a component of the outer membrane of gram-negative bacteria and serves as an endotoxin by provoking a robust innate immune response [15]. In addition to a peripheral response, intraperitoneal injections of LPS also result in cytokine elevations within the brain that include IL-1β, IL-6 and TNF-α [3, 16]. Systemic LPS administration in this way, has variously been reported to be neuroprotective [6-9], or neurotoxic [3, 17-19].
Experimental nerve injuries (axotomy) at the nerve root-CNS junction [20] serve as an effective model to produce motoneuronal death in adult animals, and consequently is used to study neuronal rescue factors [21-24]. Johnson et al [25] found that 84% of adult rat facial motoneurones die 28 days following facial nerve avulsion whereas only 22% die following nerve crush [26]. This makes: (i) nerve avulsion, the ideal severe injury-model; (ii) nerve crush, the ideal mild injury-model and (iii) 28 days post-axotomy as the ideal time-point to yield maximum motoneuronal death in both injury models and therefore to study the effects of interventions on 4
motoneuronal survival. Both these nerve injury models lend themselves to assess if LPS priming (at a pre-determined dose and time) will protect the motoneurones or make them more vulnerable to either mild or severe injuries (nerve crush and nerve avulsion respectively). This will provide insight into whether inflammation, in the form of LPS priming, can promote or prevent motoneuronal survival. Therefore, in this study we have used a well-established method of elevating inflammatory levels (intraperitoneal LPS) and a well-established model of neuronal survival (facial nerve crush or avulsion) to determine whether elevation of inflammatory levels just prior to nerve injury promotes adult facial motoneuronal survival.
2 RESULTS
24 hours after the i.p. administration of 0.5mg/kg of LPS, levels of pro-inflammatory brainstem cytokines were increased compared to adult controls (Figure 1). This is evidenced by increased cytokine levels of IL-1β (Adult: 189 ± 3 vs. LPS: 589 ± 30 vs. Ageing: 2293 ± 96); IL-5 (Adult:180 ± 11 vs. LPS: 438 ± 62 vs. Ageing: 311 ± 12) and IL-12p70 (Adult: 286 ± 34 vs. LPS: 626 ± 57 vs. Ageing: 764 ± 30). Values are given as mean ± SEM in pg/mL. This dose and administration time yielded the highest correlation of 62% (from calculations of 2 point moving average trend-lines) of cytokines with the previously published ageing cytokine profile [27]. There were no significant differences in general exploratory behaviour between the LPS administered animals, normal adults and normal ageing animals, therefore we concluded that no significant LPS-sickness behaviour was seen in the LPS-administered animals (Figure 2). However, significant exploratory behaviour variations were seen in between time-points with the 5
0.1mg/kg (4h and 24h) and 1.0 mg/kg (2h and 4h) dose administrations. As a result, the 0.5mg/kg dose was again considered optimal for further study, as the exploratory behaviour of animals, was least affected by the varying administration times with only this dose. As seen in Figure 3, nerve avulsion only resulted in 23% motoneuronal survival (p<0.01) whereas LPS priming before nerve avulsion resulted in 68% motoneuronal survival (p<0.01). Nerve crush was associated with 86% motoneuronal survival as published previously (p<0.01). Saline (94% survival) and LPS (97% survival) priming before nerve crush had no significant effect on motoneuronal survival compared to nerve crush alone.
3 DISCUSSION 3.1 Motoneurone survival
We report that an acute LPS priming in adult male rats almost triples the survival of avulsed facial motoneurones. We have also previously shown that ageing motoneurones in male rats are less likely to die following avulsion than those of adult rats [14]. Neuronal loss in ageing is not mirrored by neuronal loss in diseases such as Motor Neuron Disease (MND). Rather, we suggest that sporadic MND as a disease predominantly targeting the older demographic, needs to be studied in the context of directly aligned older motoneurones rather than younger motoneurones that have different survival requirements. While we are yet to get data for the ageing cytokine profile of male rats, the acute changes induced by LPS mimics some of the cytokine changes in ageing female rats [28]. Notwithstanding gender effects noted by studies indicating that males have a greater innate immune response [29, 30], this result tempts the speculation that a similar process occurs in ageing males. Prior administration of low doses of LPS have previously been 6
found to provide neuroprotection in experimental models of stroke [6] and traumatic brain injury [7-9]. Notwithstanding the uncertainty about how systemic LPS from intraperitoneal administration signals cells in the Central Nervous System without crossing the blood brain barrier [7], recent studies propose inflammatory priming of central nervous system neurons to be similar to the concept of ‘conditioning lesions’ in the peripheral nervous system. In one version of this concept, neuronal support cells are primed by the initial “conditioning lesion” and therefore have an immediate protective response and better repair capacity to any subsequent injury [31]. In line with this, several studies have reported perineuronal gliosis surrounding surviving neurones after experimental injury and in neurodegenerative diseases [32, 33]. This would support the idea of conditioning lesions, implicating astrocytes and microglia as the support cells mediating the neuroprotective effects, as an endotoxin/inflammatory agent (such as LPS) exerting ionotropic neuroprotective effects on widespread vulnerable motor neurones seems unlikely. This focus on uninjured neuroglial cells as potential determinants of neuronal survival represents an important shift in research emphasis that could contribute greatly to underlying mechanisms of neurodegeneration.
Pioneering studies in this field mainly reported increased gliosis around specific injured motoneurones with little mention of their association with immune mechanisms [24, 34, 35]. In the aged brain, the microglia are thought to be inherently primed [43] and have markedly increased pro-inflammatory cytokine secretion compared to young (unprimed) microglia [44]. More recently, increased gliosis around injured neurones have been hypothesised to relate to a change in the local inflammatory environment [36, 37] that affects the survival of both injured and surrounding motoneurones [38]. Supporting this theory, IL-10 expression by the astrocytes
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of transgenic mice has been shown to reduce axotomy-induced facial motoneurone death [10]. LPS-induced inflammation has been shown to be mediated by the binding of Toll-like receptor 4 on microglia [39-42]. Metabotropic effects of LPS could likely explain activation of microglia to secrete cytokines that are capable of synergising with astrocytes to protect neurones. Whether the neuroprotection provided by systemic LPS in this study involved increased activity of microglia is unknown. However, the current results have now revealed that glial activity will be worth pursuing and analysing only within the context of a severe-injury model (avulsion) to elucidate any glia-mediated neuroprotection. Microglia serve several neuroprotective functions, such as; phagocytosis of cellular debris and secretion of neurotrophic factors – both of which would be crucial in the event of a severe avulsion injury [12]. There is a large body of work characterising inflammation and its effect in neurodegeneration, especially in genetic experimental ALS mouse models (Yoshihara et al., 2002, Nguyen et al., 2004, Godbout et al., 2005). Certainly, cytokines for long have been thought as initiators of acute neurodegeneration by the activation of various apoptotic genes and caspases, depending on the insult (e.g. trauma, excitotoxicity). On the contrary, some studies indicate that acute neuroinflammation slows disease progression at earlystages, and also that chronic neuroinflammation accelerates chronic neurodegeneration (Beers et al., 2011, Moser et al., 2013). The studies pinpointing the neurodegenerative effects of neuroinflammation, approach their investigations in the context of microglia and astrocytes.
Accordingly Brown and Neher (2010) argue that pathogens along with neuronal- and vascular damage lead to inflammatory cytokine-release that encourage microglia to phagocytose all damaged neurones. However, more recent studies illustrated that microglia-associated neurodegeneration was attributed to a reduction in their phagocytic capacity rather than their
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presence (Xavier et al., 2014). This aligns with older studies that have shown microglia to be selective for the particular motoneurones they phagocytose to ensure that the CNS connectome is optimally reorganised, in the event of an injury (Kerns and Hinsman, 1973). Accordingly, impaired phagocytosis that is seen in a form of familial MND (fMND) is associated with the mutation in a gene responsible for actin dynamics in phagocytosis. In this way, mutated Profilin 1 results in impaired phagosome formation and dysfunctional phagocytosis (Petkau and Leavitt, 2014, Radford et al., 2015).
In a similar manner, diseased or dysfunctional astrocytes have been implicated in MND progression via neuroinflammation. In Chen et al. (2015), glial progenitors were derived from fMND patients’ induced pluripotent stem cells (iPSC) and grafted into mice. This resulted in acute motoneuronal degeneration and motor deficits compared to control mice. Similarly, iPSCderived astrocytes from healthy individuals transplanted into the lumbar spinal cord prolonged the lifespan of SOD1 mice (Kondo et al., 2014). Taken together, this may indicate that inflammatory gliosis is not always present in a neurodegenerative capacity. We suggest that the glial phenotype present in neuroprotective situations needs to be elucidated to then distinguish any potential variances in acute or chronic neurodegeneration.
3.2 Inflammatory cytokine changes While the acute LPS administration in this study did not reproduce the whole ageing cytokine profile, the LPS dose yielded higher levels of IL-1β, IL-5 and IL-12p70 in the adults akin to the ageing profile, where motoneuronal survival has been reportedly increased [28], as paralleled by this study. IL-1β is produced by microglia and astrocytes, is pro-inflammatory in function and has been shown to protect against oxidative stress [45]. IL-5 is anti-inflammatory and known to 9
have anti-apoptotic effects via C-Myc and the JAK cascade pathways [46, 47]. IL-12 is also produced by microglia and mainly serves as a pro-inflammatory amplifier by encouraging the production of a range of other pro-inflammatory cytokines. Recently, lower levels of IL-1β were reported in the cerebrospinal fluid (CSF) of MNDpatients where motoneurones degenerate, eventually causing fatal respiratory failure [48]. Lower CSF levels of IL-5 [49] and IL-12 (Rentzos et al., 2010) were also reported in MND patients. Albeit, inhibition of a different subunit of IL-12 known as IL-12p40 (compared to IL-12p70 studied here) has been shown to ameliorate neurodegenerative pathology in an experimental model of Alzheimer’s mice [50].
A few limitations of this study cannot be discounted. For example, the effects of experimental inflammation through LPS on motoneurones needs to be validated with alternative methodologies that only focus on intracellular expression of cytokines rather than both extra- and intra-cellular expression as assessed in our homogenised tissue samples. This is evidenced by Ford and Rowe’s [51] inability to detect a cytokine such as IL-12 in the CSF of MND patients, using their assay method. Additionally, unravelling the specific role of glial cells and the collective in-vivo effect of numerous cytokines working together (rather than just 12 cytokines as studied here) requires continuing studies and larger sample sizes to ensure that the number of variables studied do not out-number the samples studied. Caution should also be exercised when extrapolating cytokine concentrations from a specific region of the CNS to the rest of the CNS, especially when attempting to use this model of experimental inflammation to mimic ageing animals. We noted that different regions within the CNS have different cytokine profiles when analysed in the exact same LPS time- and dose- response studies, as seen in the temporal lobe (Figure 4) compared to the brainstem (Figure 1). Out of the 12 cytokines analysed in the
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temporal lobe, only IL-5, IL-6 and IL12p70 are present in LPS-administered animals to similar levels seen in normal adult and ageing animals (Figure 4). However, this did not influence our current study design as only the cytokine profile of the brainstem was of interest to us, given that the facial motoneurones that we studied are located within the brainstem. We report that acute LPS priming of the innate immune system neuroprotects adult facial motoneurones that would otherwise be vulnerable to degeneration from severe, but not mild, injury. We parallel this with previous work showing that elevated inflammatory levels in ageing rats are associated with increased survival of ageing motoneurones after injury. Taken together, this suggests that elevated inflammatory levels may be neuroprotective for motoneurones.
4 EXPERIMENTAL PROCEDURE 4.1 Optimisation of LPS dose and time of administration prior to nerve injury studies
Twenty-seven adult (3-month-old), male Sprague-Dawley rats were used in order to determine optimal LPS administration for subsequent nerve injury studies that was consistent with elevated cytokine levels but minimal sickness behaviour. To prevent selection bias, the simple randomisation criteria was used and animals were randomly allocated numbers and then further allocated to groups based on an online random-number-generator. As a dose- and time- response study, the elevation of inflammatory cytokine levels were deemed “optimal” based on the best correlation with the normal ageing inflammatory cytokine profile [27]. Rats were divided into 9 groups (n = 3) and given an intraperitoneal injection of 0.01, 0.1, or 0.5mg/kg LPS in sterile saline proportional to their weights. Rats were assayed at 2, 4 or 24 hours upon deep anaesthesia with isofluorane and transcardially perfused with saline. The brainstem and temporal lobe tissue 11
was removed and snap frozen in liquid nitrogen. Frozen samples were homogenised in lysis buffer made up of 0.1M phosphate buffered saline (PBS), Triton-X and protease inhibitors (Roche, cOmplete tablets) and the BioRad DC Protein Assay kit (Lowry method) was used to determine the total amount of protein in each sample as per manufacturer’s instructions (BioRad, New South Wales, Australia). A Bio-Plex Pro Rat 12 Plex cytokine assay kit (BioRad, New South Wales, Australia) was used to measure the concentration (pg/mL) of twelve cytokines within each sample (Interleukins 1α, 1β, 2, 4, 5, 6, 10, 12, and 13; Granulocyte Macrophage Colony Stimulating Factor, Interferon γ and Tumour Necrosis Factor α). Samples were loaded onto a 96-well plate in triplicates. Briefly, in this assay, multiple antibodies are anchored to magnetic beads which then anchor the respective antigens, allowing it to be bound by a biotinlabelled detection antibody that is fluorescently labelled with a phycoerythrin reporter molecule. A Magpix Multiplex Reader (BioRad, New South Wales) was used to determine protein levels by calibrating experimental data against standard curves for all 12 cytokines.
4.2 Open-field exploratory behaviour The open field test was used as a measure of social-exploratory behaviour and general activity to ascertain any LPS-induced sickness behaviour [52]. Visual monitoring and clinical record sheetscoring also contributed to this process. A large square box acted as the “open field” and rats were placed in the centre of the “open field” marked out by a circle. Movement was then recorded for a period of 5 minutes as the outcome (total distance travelled in metres). To ensure consistency between groups, the colour and texture of the open field box, lighting, temperature, ambient noise and olfactory cues were all controlled for. The “Stoelting ANY-maze” software was used as the tracking system that automated this functional test. The operator analysing the rats’ exploratory behaviour in the open-field functional test was blind for (i) treatment and (ii) 12
phenotype as both nerve crushes and nerve avulsions phenotypically present as a surgical scar behind the ear & paralysis of the vibrissae. Thus, there was no clear difference on gross observation to introduce operator-bias.
4.3 Facial nerve injury and motoneurone counts Twenty-four adult (3-month-old), male Sprague-Dawley rats were used. The experimental plan is detailed in Table 1. Briefly, rats were randomly divided into 4 groups and given an i.p. injection of either LPS (0.5 mg/kg) or saline. Twenty-four hours following LPS injection, rats were surgically anaesthetised and the right facial nerve was injured at the stylo mastoid foramen by either nerve-crush or nerve-avulsion. Details of the nerve injury surgical technique have been reported previously [14]. Data for non-operated rats, facial nerve avulsion only or facial nerve crush only was used from a previous study [25]. One month after surgery, rats were deeply anaesthetized and perfusion-fixed transcardially with 4% paraformaldehyde in 0.1M phosphate buffer. The total numbers of facial motoneurones in the right (injured/operated) and left (uninjured/non-operated) facial nuclei were determined using an optical disector method modified for use in the confocal microscope [26] . Briefly, the brainstem was then sectioned serially using a Vibratome and the section series containing the facial nucleus identified. Sections were randomly and systematically sampled and stained with the fluorescent dye YOYO-1 Iodide (1:1000, Molecular Probes Y3601, Australia) to determine the number of motoneurones in a merged image of two optical sections 10µm apart in Z-step (Figure 5). The mean number of motoneurones was calculated using 2 to 3 counting frames per nucleus and 3 to 4 sections per rat. This allows the total number of motoneurones in the counting frames of 90 000 µm2 to be calculated. The volume of each facial nucleus was determined by the
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Cavalieri principle and then factored into the motoneurone counts for accuracy [53]. This stereological technique is an established and robust counting method that avoids the risk of counting the same neurone twice (Mayhew and Gundersen, 1996, Howell et al., 2002). Results are expressed as a percentage of the number of motoneurones counted in the right (injured/operated) side over the left (uninjured/non-operated) side.
4.4 Statistical Analysis Mean ± SEM values are reported. As per normality tests (D’Agostino & Pearson; Shapiro-Wilk & Q-Q plots), our data was not normally distributed and so non-parametric tests were chosen for statistical analysis. To avoid inflation of Type 1 error rate, the Kruskal-Wallis test was used to determine overall statistical significance of mean motor neuron numbers and mean cytokine changes along with Dunn’s post-hoc analysis. For pair-wise comparisons of data generated from the current study, the non-parametric Mann-Whitney U test was used.
All experiments were approved by the University of Adelaide Animal Ethics Committee (M-572013) which observes national and international guidelines on the care and use of animals for scientific purposes [54].
ACKNOWLEDGEMENTS The study was funded by the University of Adelaide and confocal microscopy was conducted in Adelaide Microscopy.
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[40] Y.C. Lu, W.C. Yeh, P.S. Ohashi, LPS/TLR4 signal transduction pathway, Cytokine, 42 (2008) 145-151. [41] D.Y. Jung, H. Lee, B.Y. Jung, J. Ock, M.S. Lee, W.H. Lee, K. Suk, TLR4, but not TLR2, signals autoregulatory apoptosis of cultured microglia: a critical role of IFN-beta as a decision maker, J Immunol, 174 (2005) 6467-6476. [42] S. Chakravarty, M. Herkenham, Toll-like receptor 4 on nonhematopoietic cells sustains CNS inflammation during endotoxemia, independent of systemic cytokines, J Neurosci, 25 (2005) 1788-1796. [43] A. Lourbopoulos, A. Erturk, F. Hellal, Microglia in action: how aging and injury can change the brain's guardians, Front. Cell. Neurosci., 9 (2015) 54. [44] E.G. Njie, E. Boelen, F.R. Stassen, H.W. Steinbusch, D.R. Borchelt, W.J. Streit, Ex vivo cultures of microglia from young and aged rodent brain reveal age-related changes in microglial function, Neurobiology of aging, 33 (2012) 195.e191-112. [45] Y. He, N.A. Jackman, T.L. Thorn, V.E. Vought, S.J. Hewett, Interleukin-1beta protects astrocytes against oxidant-induced injury via an NF-kappaB-dependent upregulation of glutathione synthesis, Glia, 63 (2015) 1568-1580. [46] W.H. Lee, F.H. Liu, J.Y. Lin, S.Y. Huang, H. Lin, W.J. Liao, H.M. Huang, JAK pathway induction of c-Myc critical to IL-5 stimulation of cell proliferation and inhibition of apoptosis, J. Cell. Biochem., 106 (2009) 929-936. [47] K. Takatsu, Interleukin-5, Curr. Opin. Immunol., 4 (1992) 299-306. [48] A.L. Lind, D. Wu, E. Freyhult, C. Bodolea, T. Ekegren, A. Larsson, M.G. Gustafsson, L. Katila, J. Bergquist, T. Gordh, U. Landegren, M. Kamali-Moghaddam, A Multiplex Protein Panel Applied to Cerebrospinal Fluid Reveals Three New Biomarker Candidates in ALS but None in Neuropathic Pain Patients, PLoS One, 11 (2016) e0149821. [49] J. Ehrhart, A.J. Smith, N. Kuzmin-Nichols, T.A. Zesiewicz, I. Jahan, R.D. Shytle, S.H. Kim, C.D. Sanberg, T.H. Vu, C.L. Gooch, P.R. Sanberg, S. Garbuzova-Davis, Humoral factors in ALS patients during disease progression, J Neuroinflammation, 12 (2015) 127. [50] M.S. Tan, J.T. Yu, T. Jiang, X.C. Zhu, H.S. Guan, L. Tan, IL12/23 p40 inhibition ameliorates Alzheimer's disease-associated neuropathology and spatial memory in SAMP8 mice, Journal of Alzheimer's disease : JAD, 38 (2014) 633-646. [51] L.S. Ford, D.B. Rowe, Interleukin-12 and interferon-γ are not detectable in the cerebrospinal fluid of patients with amyotrophic lateral sclerosis, Amyotrophic Lateral Sclerosis & Other Motor Neuron Disorders, 5 (2004) 118-120. [52] T.D. Gould, D.T. Dao, C.E. Kovacsics, The Open Field Test, 42 (2009) 1-20. [53] H.J. Gundersen, E.B. Jensen, The efficiency of systematic sampling in stereology and its prediction, Journal of Microscopy, 147 (1987) 229-263. [54] NHMRC, Australian code for the care and use of animals for scientific purposes, Australian Government, Canberra, 2013.
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FIGURE/TABLE LEGENDS Figure 1 Comparative brainstem cytokine profiles for adult (3 months old), ageing (24 months old) and LPS treated rats as time- and dose- response studies. The optimal time and dose was assessed as the closest match to the ageing cytokine profile. The 0.5mg/kg dose and 24 hour time-point was chosen with a 62% overall correlation with the ageing cytokine profile.
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Figure 2 General exploratory behaviour measured by the open-field functional tests. While differences between LPS-administered animals were found, no significant differences were noted between normal adults, aging animals and the LPS-administered animals. * denotes p<0.05 and *** denotes p<0.001.
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Figure 3 Mean number of motoneurones from stereological counts expressed as a percentage of motoneurones counted in the injured (operated) facial nucleus over uninjured (non-operated internal control) facial nucleus. ** denotes p<0.01.
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Figure 4 Comparative temporal lobe cytokine profiles for adult, ageing and LPS treated rats as time- and dose- response studies.
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Table 1
Experimental design indicating comparison data for age and gender matched Sprague-Dawley rats, taken from previously published studies by the authors. The effect of inflammation on motoneurones was assessed in this study by priming the motoneurones with LPS prior to a less severe (crush) injury and a more severe (avulsion) injury. Saline priming animals served as an internal control. The number of rats in each group is given in parentheses.
Figure 5 Confocal microscopy images of the facial nucleus stained with fluorescent YOYO-1 iodide. For the purposes of stereological analysis, motoneurones (assessed as being alpha motoneurones) were counted if they met the criteria of being 30 µm or more in diameter (seen in white circles). Two pseudo-coloured images (red or green) 10 µm apart in Z-step are taken. The merged images (seen in this figure) serves as the counting frame. Only non-overlapping motoneurones (in green colour) are counted to avoid counting the same neuron twice, as is standard stereological practice. Here, more motoneurones are seen in the “Non-operated” image, compared to the “Avulsion-only” image. Whereas, more motoneurones are seen in the “LPS + Avulsion” image compared to the “Avulsion-only” image. Also seen in these images are Central Nervous System glial cells (white arrows). 22
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HIGHLIGHTS
Experimental inflammation can increase rat cytokines without sickness behaviour.
Increased inflammatory cytokines neuroprotect severely injured motoneurones.
This neuroprotection is not seen in mildly injured adult rat facial motoneurones.
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S0006-8993(18)30309-3 https://doi.org/10.1016/j.brainres.2018.05.039 BRES 45822
To appear in:
Brain Research
Received Date: Revised Date: Accepted Date:
3 January 2018 22 May 2018 25 May 2018
Please cite this article as: V. Katharesan, S. Deery, I.P. Johnson, Neuroprotective effect of acute prior inflammation with lipopolysaccharide for adult male rat facial motoneurones, Brain Research (2018), doi: https://doi.org/10.1016/ j.brainres.2018.05.039
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Viythia Katharesan, Shane Deery, and Ian P Johnson* Discipline of Anatomy and Pathology The University of Adelaide Australia SA5005 Neuroprotective effect of acute prior inflammation with lipopolysaccharide for adult male rat facial motoneurones
*Corresponding author: Email: [email protected] Telephone: +61 8 8313 4526
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ABBREVIATIONS CLSM: Confocal laser scanning microscope GM-CSF: Granulocyte Macrophage Colony Stimulating Factor LPS: Lipopolysaccharide IFN-γ: Interferon γ IL: Interleukin TNF-α: Tumour Necrosis Factor α MND: Motor Neuron Disease CSF: Cerebrospinal fluid
ABSTRACT Increases in inflammatory cytokines are reported to have both neuroprotective and neurotoxic effects depending on the type and age of neurones studied. This study aimed to determine the effect of experimental inflammation induced by Lipopolysaccharide (LPS) on the survival of injured male adult rat facial motoneurones. Time- and dose- response studies were done to optimise the LPS administration time and dose, to best correlate with inflammatory levels previously reported for aged rats. 12 cytokines were assayed through multiplex analysis. 24 hours after intraperitoneal injection of 0.5mg/kg Lipopolysaccharide in rats, IL-1β, IL-5 and IL12p70 levels were elevated, with no observed LPS-associated sickness behaviour. In other groups of 5-6 adult rats, the facial nerve was either crushed (as mild injury) or avulsed (as severe
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injury) after the LPS priming injection. Stereology revealed that most motoneurones survived 28 days after nerve crush only and LPS- or saline-priming preceding nerve crush. Most motoneurones died following nerve avulsion only, whereas over half survived when LPSpriming preceded nerve avulsion. We suggest that elevated levels of experimental inflammation are neuroprotective for severely injured adult male rat facial motoneurones.
KEY WORDS Neuroinflammation, Cytokines, Stereology, Motor neuron, Rescue, Facial Nerve
1 INTRODUCTION
Inflammatory status has a major impact on neuronal survival, but whether it promotes or prevents neuronal degeneration is unclear. Elevated inflammatory levels due to challenge by pathogens or by trauma have been associated with neuronal dysfunction and degeneration in both developing- and adult- animals [1-3]. Increased inflammation with ageing animals has been implicated in the pathogenesis of age-related neurodegenerative disease [4, 5]. In contrast, increased inflammatory levels after LPS priming (intraperitoneally) have been found to provide neuroprotection in experimental models of stroke [6] and traumatic brain injury [7-9]. Furthermore, local expression of IL-10 by astrocytes increases the survival of axotomised/injured motoneurones [10]. In age-related neurodegenerative diseases, there is increasing evidence to show that neuroinflammation may be resultant, rather than causative, of disease [11], and it has been proposed that microglial activation with neuroinflammaging may in fact be neuroprotective [4, 12]. Part of this confusion about the role of inflammation in neurodegeneration and neuronal 3
survival may be due to the difficulty of comparing studies using varying models of inflammation and the inherent limitation of studying neuronal death in ageing animals, which are very difficult to obtain and maintain. Sustaining experimental geriatric animals for long periods, while keeping in line with animal ethics, is difficult [13] and unlike their young counterparts, ageing animals’ motoneurones can be more robust and resistant to experimental injury [14]. Thus, there tends to be a preference to work on young animals, which are easier to maintain and in which, it is easier to provoke rapid and substantial neuronal death. This inevitably lends young animals as a productive, but unsuited model that leaves extrapolation of data non-transferable to neurodegenerative conditions in which the ageing demographic, and therefore ageing neurones are affected.
Lipopolysaccharide is a component of the outer membrane of gram-negative bacteria and serves as an endotoxin by provoking a robust innate immune response [15]. In addition to a peripheral response, intraperitoneal injections of LPS also result in cytokine elevations within the brain that include IL-1β, IL-6 and TNF-α [3, 16]. Systemic LPS administration in this way, has variously been reported to be neuroprotective [6-9], or neurotoxic [3, 17-19].
Experimental nerve injuries (axotomy) at the nerve root-CNS junction [20] serve as an effective model to produce motoneuronal death in adult animals, and consequently is used to study neuronal rescue factors [21-24]. Johnson et al [25] found that 84% of adult rat facial motoneurones die 28 days following facial nerve avulsion whereas only 22% die following nerve crush [26]. This makes: (i) nerve avulsion, the ideal severe injury-model; (ii) nerve crush, the ideal mild injury-model and (iii) 28 days post-axotomy as the ideal time-point to yield maximum motoneuronal death in both injury models and therefore to study the effects of interventions on 4
motoneuronal survival. Both these nerve injury models lend themselves to assess if LPS priming (at a pre-determined dose and time) will protect the motoneurones or make them more vulnerable to either mild or severe injuries (nerve crush and nerve avulsion respectively). This will provide insight into whether inflammation, in the form of LPS priming, can promote or prevent motoneuronal survival. Therefore, in this study we have used a well-established method of elevating inflammatory levels (intraperitoneal LPS) and a well-established model of neuronal survival (facial nerve crush or avulsion) to determine whether elevation of inflammatory levels just prior to nerve injury promotes adult facial motoneuronal survival.
2 RESULTS
24 hours after the i.p. administration of 0.5mg/kg of LPS, levels of pro-inflammatory brainstem cytokines were increased compared to adult controls (Figure 1). This is evidenced by increased cytokine levels of IL-1β (Adult: 189 ± 3 vs. LPS: 589 ± 30 vs. Ageing: 2293 ± 96); IL-5 (Adult:180 ± 11 vs. LPS: 438 ± 62 vs. Ageing: 311 ± 12) and IL-12p70 (Adult: 286 ± 34 vs. LPS: 626 ± 57 vs. Ageing: 764 ± 30). Values are given as mean ± SEM in pg/mL. This dose and administration time yielded the highest correlation of 62% (from calculations of 2 point moving average trend-lines) of cytokines with the previously published ageing cytokine profile [27]. There were no significant differences in general exploratory behaviour between the LPS administered animals, normal adults and normal ageing animals, therefore we concluded that no significant LPS-sickness behaviour was seen in the LPS-administered animals (Figure 2). However, significant exploratory behaviour variations were seen in between time-points with the 5
0.1mg/kg (4h and 24h) and 1.0 mg/kg (2h and 4h) dose administrations. As a result, the 0.5mg/kg dose was again considered optimal for further study, as the exploratory behaviour of animals, was least affected by the varying administration times with only this dose. As seen in Figure 3, nerve avulsion only resulted in 23% motoneuronal survival (p<0.01) whereas LPS priming before nerve avulsion resulted in 68% motoneuronal survival (p<0.01). Nerve crush was associated with 86% motoneuronal survival as published previously (p<0.01). Saline (94% survival) and LPS (97% survival) priming before nerve crush had no significant effect on motoneuronal survival compared to nerve crush alone.
3 DISCUSSION 3.1 Motoneurone survival
We report that an acute LPS priming in adult male rats almost triples the survival of avulsed facial motoneurones. We have also previously shown that ageing motoneurones in male rats are less likely to die following avulsion than those of adult rats [14]. Neuronal loss in ageing is not mirrored by neuronal loss in diseases such as Motor Neuron Disease (MND). Rather, we suggest that sporadic MND as a disease predominantly targeting the older demographic, needs to be studied in the context of directly aligned older motoneurones rather than younger motoneurones that have different survival requirements. While we are yet to get data for the ageing cytokine profile of male rats, the acute changes induced by LPS mimics some of the cytokine changes in ageing female rats [28]. Notwithstanding gender effects noted by studies indicating that males have a greater innate immune response [29, 30], this result tempts the speculation that a similar process occurs in ageing males. Prior administration of low doses of LPS have previously been 6
found to provide neuroprotection in experimental models of stroke [6] and traumatic brain injury [7-9]. Notwithstanding the uncertainty about how systemic LPS from intraperitoneal administration signals cells in the Central Nervous System without crossing the blood brain barrier [7], recent studies propose inflammatory priming of central nervous system neurons to be similar to the concept of ‘conditioning lesions’ in the peripheral nervous system. In one version of this concept, neuronal support cells are primed by the initial “conditioning lesion” and therefore have an immediate protective response and better repair capacity to any subsequent injury [31]. In line with this, several studies have reported perineuronal gliosis surrounding surviving neurones after experimental injury and in neurodegenerative diseases [32, 33]. This would support the idea of conditioning lesions, implicating astrocytes and microglia as the support cells mediating the neuroprotective effects, as an endotoxin/inflammatory agent (such as LPS) exerting ionotropic neuroprotective effects on widespread vulnerable motor neurones seems unlikely. This focus on uninjured neuroglial cells as potential determinants of neuronal survival represents an important shift in research emphasis that could contribute greatly to underlying mechanisms of neurodegeneration.
Pioneering studies in this field mainly reported increased gliosis around specific injured motoneurones with little mention of their association with immune mechanisms [24, 34, 35]. In the aged brain, the microglia are thought to be inherently primed [43] and have markedly increased pro-inflammatory cytokine secretion compared to young (unprimed) microglia [44]. More recently, increased gliosis around injured neurones have been hypothesised to relate to a change in the local inflammatory environment [36, 37] that affects the survival of both injured and surrounding motoneurones [38]. Supporting this theory, IL-10 expression by the astrocytes
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of transgenic mice has been shown to reduce axotomy-induced facial motoneurone death [10]. LPS-induced inflammation has been shown to be mediated by the binding of Toll-like receptor 4 on microglia [39-42]. Metabotropic effects of LPS could likely explain activation of microglia to secrete cytokines that are capable of synergising with astrocytes to protect neurones. Whether the neuroprotection provided by systemic LPS in this study involved increased activity of microglia is unknown. However, the current results have now revealed that glial activity will be worth pursuing and analysing only within the context of a severe-injury model (avulsion) to elucidate any glia-mediated neuroprotection. Microglia serve several neuroprotective functions, such as; phagocytosis of cellular debris and secretion of neurotrophic factors – both of which would be crucial in the event of a severe avulsion injury [12]. There is a large body of work characterising inflammation and its effect in neurodegeneration, especially in genetic experimental ALS mouse models (Yoshihara et al., 2002, Nguyen et al., 2004, Godbout et al., 2005). Certainly, cytokines for long have been thought as initiators of acute neurodegeneration by the activation of various apoptotic genes and caspases, depending on the insult (e.g. trauma, excitotoxicity). On the contrary, some studies indicate that acute neuroinflammation slows disease progression at earlystages, and also that chronic neuroinflammation accelerates chronic neurodegeneration (Beers et al., 2011, Moser et al., 2013). The studies pinpointing the neurodegenerative effects of neuroinflammation, approach their investigations in the context of microglia and astrocytes.
Accordingly Brown and Neher (2010) argue that pathogens along with neuronal- and vascular damage lead to inflammatory cytokine-release that encourage microglia to phagocytose all damaged neurones. However, more recent studies illustrated that microglia-associated neurodegeneration was attributed to a reduction in their phagocytic capacity rather than their
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presence (Xavier et al., 2014). This aligns with older studies that have shown microglia to be selective for the particular motoneurones they phagocytose to ensure that the CNS connectome is optimally reorganised, in the event of an injury (Kerns and Hinsman, 1973). Accordingly, impaired phagocytosis that is seen in a form of familial MND (fMND) is associated with the mutation in a gene responsible for actin dynamics in phagocytosis. In this way, mutated Profilin 1 results in impaired phagosome formation and dysfunctional phagocytosis (Petkau and Leavitt, 2014, Radford et al., 2015).
In a similar manner, diseased or dysfunctional astrocytes have been implicated in MND progression via neuroinflammation. In Chen et al. (2015), glial progenitors were derived from fMND patients’ induced pluripotent stem cells (iPSC) and grafted into mice. This resulted in acute motoneuronal degeneration and motor deficits compared to control mice. Similarly, iPSCderived astrocytes from healthy individuals transplanted into the lumbar spinal cord prolonged the lifespan of SOD1 mice (Kondo et al., 2014). Taken together, this may indicate that inflammatory gliosis is not always present in a neurodegenerative capacity. We suggest that the glial phenotype present in neuroprotective situations needs to be elucidated to then distinguish any potential variances in acute or chronic neurodegeneration.
3.2 Inflammatory cytokine changes While the acute LPS administration in this study did not reproduce the whole ageing cytokine profile, the LPS dose yielded higher levels of IL-1β, IL-5 and IL-12p70 in the adults akin to the ageing profile, where motoneuronal survival has been reportedly increased [28], as paralleled by this study. IL-1β is produced by microglia and astrocytes, is pro-inflammatory in function and has been shown to protect against oxidative stress [45]. IL-5 is anti-inflammatory and known to 9
have anti-apoptotic effects via C-Myc and the JAK cascade pathways [46, 47]. IL-12 is also produced by microglia and mainly serves as a pro-inflammatory amplifier by encouraging the production of a range of other pro-inflammatory cytokines. Recently, lower levels of IL-1β were reported in the cerebrospinal fluid (CSF) of MNDpatients where motoneurones degenerate, eventually causing fatal respiratory failure [48]. Lower CSF levels of IL-5 [49] and IL-12 (Rentzos et al., 2010) were also reported in MND patients. Albeit, inhibition of a different subunit of IL-12 known as IL-12p40 (compared to IL-12p70 studied here) has been shown to ameliorate neurodegenerative pathology in an experimental model of Alzheimer’s mice [50].
A few limitations of this study cannot be discounted. For example, the effects of experimental inflammation through LPS on motoneurones needs to be validated with alternative methodologies that only focus on intracellular expression of cytokines rather than both extra- and intra-cellular expression as assessed in our homogenised tissue samples. This is evidenced by Ford and Rowe’s [51] inability to detect a cytokine such as IL-12 in the CSF of MND patients, using their assay method. Additionally, unravelling the specific role of glial cells and the collective in-vivo effect of numerous cytokines working together (rather than just 12 cytokines as studied here) requires continuing studies and larger sample sizes to ensure that the number of variables studied do not out-number the samples studied. Caution should also be exercised when extrapolating cytokine concentrations from a specific region of the CNS to the rest of the CNS, especially when attempting to use this model of experimental inflammation to mimic ageing animals. We noted that different regions within the CNS have different cytokine profiles when analysed in the exact same LPS time- and dose- response studies, as seen in the temporal lobe (Figure 4) compared to the brainstem (Figure 1). Out of the 12 cytokines analysed in the
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temporal lobe, only IL-5, IL-6 and IL12p70 are present in LPS-administered animals to similar levels seen in normal adult and ageing animals (Figure 4). However, this did not influence our current study design as only the cytokine profile of the brainstem was of interest to us, given that the facial motoneurones that we studied are located within the brainstem. We report that acute LPS priming of the innate immune system neuroprotects adult facial motoneurones that would otherwise be vulnerable to degeneration from severe, but not mild, injury. We parallel this with previous work showing that elevated inflammatory levels in ageing rats are associated with increased survival of ageing motoneurones after injury. Taken together, this suggests that elevated inflammatory levels may be neuroprotective for motoneurones.
4 EXPERIMENTAL PROCEDURE 4.1 Optimisation of LPS dose and time of administration prior to nerve injury studies
Twenty-seven adult (3-month-old), male Sprague-Dawley rats were used in order to determine optimal LPS administration for subsequent nerve injury studies that was consistent with elevated cytokine levels but minimal sickness behaviour. To prevent selection bias, the simple randomisation criteria was used and animals were randomly allocated numbers and then further allocated to groups based on an online random-number-generator. As a dose- and time- response study, the elevation of inflammatory cytokine levels were deemed “optimal” based on the best correlation with the normal ageing inflammatory cytokine profile [27]. Rats were divided into 9 groups (n = 3) and given an intraperitoneal injection of 0.01, 0.1, or 0.5mg/kg LPS in sterile saline proportional to their weights. Rats were assayed at 2, 4 or 24 hours upon deep anaesthesia with isofluorane and transcardially perfused with saline. The brainstem and temporal lobe tissue 11
was removed and snap frozen in liquid nitrogen. Frozen samples were homogenised in lysis buffer made up of 0.1M phosphate buffered saline (PBS), Triton-X and protease inhibitors (Roche, cOmplete tablets) and the BioRad DC Protein Assay kit (Lowry method) was used to determine the total amount of protein in each sample as per manufacturer’s instructions (BioRad, New South Wales, Australia). A Bio-Plex Pro Rat 12 Plex cytokine assay kit (BioRad, New South Wales, Australia) was used to measure the concentration (pg/mL) of twelve cytokines within each sample (Interleukins 1α, 1β, 2, 4, 5, 6, 10, 12, and 13; Granulocyte Macrophage Colony Stimulating Factor, Interferon γ and Tumour Necrosis Factor α). Samples were loaded onto a 96-well plate in triplicates. Briefly, in this assay, multiple antibodies are anchored to magnetic beads which then anchor the respective antigens, allowing it to be bound by a biotinlabelled detection antibody that is fluorescently labelled with a phycoerythrin reporter molecule. A Magpix Multiplex Reader (BioRad, New South Wales) was used to determine protein levels by calibrating experimental data against standard curves for all 12 cytokines.
4.2 Open-field exploratory behaviour The open field test was used as a measure of social-exploratory behaviour and general activity to ascertain any LPS-induced sickness behaviour [52]. Visual monitoring and clinical record sheetscoring also contributed to this process. A large square box acted as the “open field” and rats were placed in the centre of the “open field” marked out by a circle. Movement was then recorded for a period of 5 minutes as the outcome (total distance travelled in metres). To ensure consistency between groups, the colour and texture of the open field box, lighting, temperature, ambient noise and olfactory cues were all controlled for. The “Stoelting ANY-maze” software was used as the tracking system that automated this functional test. The operator analysing the rats’ exploratory behaviour in the open-field functional test was blind for (i) treatment and (ii) 12
phenotype as both nerve crushes and nerve avulsions phenotypically present as a surgical scar behind the ear & paralysis of the vibrissae. Thus, there was no clear difference on gross observation to introduce operator-bias.
4.3 Facial nerve injury and motoneurone counts Twenty-four adult (3-month-old), male Sprague-Dawley rats were used. The experimental plan is detailed in Table 1. Briefly, rats were randomly divided into 4 groups and given an i.p. injection of either LPS (0.5 mg/kg) or saline. Twenty-four hours following LPS injection, rats were surgically anaesthetised and the right facial nerve was injured at the stylo mastoid foramen by either nerve-crush or nerve-avulsion. Details of the nerve injury surgical technique have been reported previously [14]. Data for non-operated rats, facial nerve avulsion only or facial nerve crush only was used from a previous study [25]. One month after surgery, rats were deeply anaesthetized and perfusion-fixed transcardially with 4% paraformaldehyde in 0.1M phosphate buffer. The total numbers of facial motoneurones in the right (injured/operated) and left (uninjured/non-operated) facial nuclei were determined using an optical disector method modified for use in the confocal microscope [26] . Briefly, the brainstem was then sectioned serially using a Vibratome and the section series containing the facial nucleus identified. Sections were randomly and systematically sampled and stained with the fluorescent dye YOYO-1 Iodide (1:1000, Molecular Probes Y3601, Australia) to determine the number of motoneurones in a merged image of two optical sections 10µm apart in Z-step (Figure 5). The mean number of motoneurones was calculated using 2 to 3 counting frames per nucleus and 3 to 4 sections per rat. This allows the total number of motoneurones in the counting frames of 90 000 µm2 to be calculated. The volume of each facial nucleus was determined by the
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Cavalieri principle and then factored into the motoneurone counts for accuracy [53]. This stereological technique is an established and robust counting method that avoids the risk of counting the same neurone twice (Mayhew and Gundersen, 1996, Howell et al., 2002). Results are expressed as a percentage of the number of motoneurones counted in the right (injured/operated) side over the left (uninjured/non-operated) side.
4.4 Statistical Analysis Mean ± SEM values are reported. As per normality tests (D’Agostino & Pearson; Shapiro-Wilk & Q-Q plots), our data was not normally distributed and so non-parametric tests were chosen for statistical analysis. To avoid inflation of Type 1 error rate, the Kruskal-Wallis test was used to determine overall statistical significance of mean motor neuron numbers and mean cytokine changes along with Dunn’s post-hoc analysis. For pair-wise comparisons of data generated from the current study, the non-parametric Mann-Whitney U test was used.
All experiments were approved by the University of Adelaide Animal Ethics Committee (M-572013) which observes national and international guidelines on the care and use of animals for scientific purposes [54].
ACKNOWLEDGEMENTS The study was funded by the University of Adelaide and confocal microscopy was conducted in Adelaide Microscopy.
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FIGURE/TABLE LEGENDS Figure 1 Comparative brainstem cytokine profiles for adult (3 months old), ageing (24 months old) and LPS treated rats as time- and dose- response studies. The optimal time and dose was assessed as the closest match to the ageing cytokine profile. The 0.5mg/kg dose and 24 hour time-point was chosen with a 62% overall correlation with the ageing cytokine profile.
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Figure 2 General exploratory behaviour measured by the open-field functional tests. While differences between LPS-administered animals were found, no significant differences were noted between normal adults, aging animals and the LPS-administered animals. * denotes p<0.05 and *** denotes p<0.001.
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Figure 3 Mean number of motoneurones from stereological counts expressed as a percentage of motoneurones counted in the injured (operated) facial nucleus over uninjured (non-operated internal control) facial nucleus. ** denotes p<0.01.
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Figure 4 Comparative temporal lobe cytokine profiles for adult, ageing and LPS treated rats as time- and dose- response studies.
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Table 1
Experimental design indicating comparison data for age and gender matched Sprague-Dawley rats, taken from previously published studies by the authors. The effect of inflammation on motoneurones was assessed in this study by priming the motoneurones with LPS prior to a less severe (crush) injury and a more severe (avulsion) injury. Saline priming animals served as an internal control. The number of rats in each group is given in parentheses.
Figure 5 Confocal microscopy images of the facial nucleus stained with fluorescent YOYO-1 iodide. For the purposes of stereological analysis, motoneurones (assessed as being alpha motoneurones) were counted if they met the criteria of being 30 µm or more in diameter (seen in white circles). Two pseudo-coloured images (red or green) 10 µm apart in Z-step are taken. The merged images (seen in this figure) serves as the counting frame. Only non-overlapping motoneurones (in green colour) are counted to avoid counting the same neuron twice, as is standard stereological practice. Here, more motoneurones are seen in the “Non-operated” image, compared to the “Avulsion-only” image. Whereas, more motoneurones are seen in the “LPS + Avulsion” image compared to the “Avulsion-only” image. Also seen in these images are Central Nervous System glial cells (white arrows). 22
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HIGHLIGHTS
Experimental inflammation can increase rat cytokines without sickness behaviour.
Increased inflammatory cytokines neuroprotect severely injured motoneurones.
This neuroprotection is not seen in mildly injured adult rat facial motoneurones.
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