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Temporal assessment of traumatic axonal injury in the rat corpus callosum and optic chiasm
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Research Report
Temporal assessment of traumatic axonal injury in the rat corpus callosum and optic chiasm Nisrine Zakaria⁎, Srinivasu Kallakuri1 , Sharath Bandaru1 , John M. Cavanaugh1 College of Engineering, Department of Biomedical Engineering, Wayne State University, Detroit, MI 48202, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
Impaired axoplasmic transport (IAT) and neurofilament compaction (NFC), two common
Accepted 22 May 2012
axonal pathology processes involved in traumatic axonal injury (TAI), have been well
Available online 28 May 2012
characterized. TAI is found clinically and in animal models in brainstem white matter (WM) tracts and in the corpus callosum (CC), optic chiasm (Och), and internal capsule. Previous
Keywords:
published quantitative studies of the time course of TAI expression induced by the
Traumatic axonal injury
Marmarou impact acceleration model have been limited to the brainstem. Accordingly, this
Corpus callosum
study assessed the extent of IAT and NFC in the CC and Och at 8 h, 28 h, 3 days and 7 days
Optic chiasm
after traumatic brain injury (TBI) induction by the Marmarou impact acceleration model.
Impaired axoplasmic transport
IAT peak density was observed at 8 h in the CC and 28 h in the Och post-TBI. NFC peak
Neurofilament compaction
density was observed at 28 h in both structures. The density of IAT and NFC decreased with
Impact acceleration model
increasing survival time in both structures. The NFC density time profile followed a similar trend in both the Och and CC, whereas the IAT density time profile was variable between the Och and CC. Furthermore, a strong linear relationship was observed between IAT and NFC in the CC but not in the Och. These findings highlight the heterogeneity of TAI as evidenced by variable IAT and NFC injury time profiles in each anatomical structure. This variability indicates the requirement of multiple markers for a comprehensive TAI evaluation and multiple targeted treatments for TAI polypathology within its therapeutic window time frame. © 2012 Elsevier B.V. All rights reserved.
1.
Introduction
Diffuse axonal injury (DAI), also referred to as traumatic axonal injury (TAI), is a well-recognized consequence of blunt head
injury (Adams et al., 1982). TAI is considered a major contributor to morbidity and mortality after traumatic brain injury (TBI) (Adams et al., 1989; Bennett et al., 1995; Gentleman et al., 1995; Povlishock and Christman, 1995; Slazinski and Johnson, 1994).
⁎ Corresponding author at: Department of Biomedical Engineering, 818 West Hancock, Wayne State University, Detroit, MI 48201, USA. Fax: + 1 313 577 8333. E-mail addresses: [email protected] (N. Zakaria), [email protected] (S. Kallakuri), [email protected] (S. Bandaru), [email protected] (J.M. Cavanaugh). Abbreviations: β-APP, beta amyloid precursor protein; CC, corpus callosum; DAI, diffuse axonal injury; IAT, impaired axoplasmic transport; IR, immunoreactive; NFC, neurofilament compaction; NF-M, neurofilament medium; Och, optic chiasm; TAI, traumatic axonal injury; TBI, traumatic brain injury; WM, white matter 1 Fax: +1 313 577 8333. 0006-8993/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2012.05.046
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TAI is produced by rapid head acceleration/deceleration during a traumatic event (Kelley et al., 2006) with consequent tension or shear on axons. Clinically, TAI has been reported to appear throughout the deep and subcortical white matter (WM) and has been noted to be predominantly common in the midline structures, including the corpus callosum (CC) and brainstem (Meythaler et al., 2001; Smith et al., 2003). In addition, vision impairment has also been reported to occur clinically following closed head injury (Perunovic et al., 2001). A post-mortem immunocytochemistry study of severely closed head injury cases revealed the presence of TAI at different sites within the optic pathways, such as the optic chiasm (Och), optic tract and optic radiations (Perunovic et al., 2001). The Marmarou impact acceleration weight-drop model has been widely used since its origination in 1994 to induce closed head TBI in rats in order to study TAI and other pathological changes. This model has been reported to induce DAI, particularly in the CC, internal capsule, optic tracts, cerebral and cerebellar peduncles and in the long tracts in the brainstem (Foda and Marmarou, 1994). Despite clinical findings of TAI in the subcortical WM and optic pathways, laboratory studies of TAI in these anatomical structures are limited, especially those using impact acceleration injury models. The majority of TAI studies using the Marmarou model have focused on the brainstem (Buki et al., 2000; Marmarou and Povlishock, 2006; Marmarou et al., 2005; Povlishock et al., 1997; Stone et al., 2000, 2001, 2004; Suehiro et al., 2001). The original histological study of the impact acceleration model by Foda and Marmarou (1994) reported that TAI does occur in the brainstem and to a lesser extent in the cortical WM. To the best of our knowledge, only five histological studies utilizing the Marmarou model have reported TAI in the cortical WM in a quantitative manner (Ding et al., 2001; Kallakuri et al., 2003; Kallakuri et al., 2012; Li et al., 2011a; Li et al., 2011b). These studies showed that it is possible to induce TAI in the CC (Kallakuri et al., 2003; Kallakuri et al., 2012; Li et al., 2011a, 2011b) and optic chiasm (Och) (Ding et al., 2001) using the Marmarou model. Because clinical studies have indicated that the CC and Och pathways are prominent areas of TAI after TBI, further laboratory studies are needed to assess the extent of TAI in these regions following closed-head TBI. The few laboratory studies that have quantified TAI in the CC (Kallakuri et al., 2003; Kallakuri et al., 2012; Li et al., 2011a, 2011b) and the Och (Ding et al., 2001) using this model are limited to studying TAI expression at 24 h post-TBI. However, a temporal assessment is lacking of TAI density in these structures following TBI induced by impact acceleration. Beta amyloid precursor protein (β-APP) immunostaining has been found valuable in the detection of TAI as early as 30–35 min after the traumatic event in rats and humans (Gorrie et al., 2002; Hortobagyi et al., 2007; Marmarou et al., 2005; Stone et al., 2001) and was used extensively as a marker of impaired axoplasmic transport (IAT) (DiLeonardi et al., 2009; Geddes et al., 2003; Gorrie et al., 2002; Hortobagyi et al., 2007; Marmarou et al., 2005; McKenzie et al., 1996; Pal et al., 2006; Stone et al., 2001; Suehiro et al., 2001). In addition to IAT, the intra-axonal cytoskeletal alteration characterized as neurofilament compaction (NFC) has been increasingly investigated by utilizing specific neurofilament medium chain (NF-M) antibodies (Czeiter et al., 2008; Stone et al., 2001; Suehiro et al., 2001). NFC was shown to be associated with either dephosphorylation or calpain-mediated
proteolysis of neurofilament sidearms (Buki et al., 1999a; Giza and Hovda, 2001; Marmarou et al., 2005). The monoclonal RMO14 antibodies specifically recognize epitopes on the NF-M rod domain that become available after dephosphorylation, enabling the detection of NFC (Trojanowski et al., 1989). IAT and NFC are two distinct processes occurring in distinct axonal populations (Stone et al., 2001). Compared to other histological methods such as silver staining, immunohistochemistry has the advantage of detecting subcellular aspects of TAI such as IAT and NFC (DiLeonardi et al., 2009; Marmarou et al., 2005; Pal et al., 2006; Stone et al., 2001; Suehiro et al., 2001). The purpose of this study was to assess the subcellular aspects of TAI density over time in two clinically relevant WM tracts. Specifically, the temporal subcellular TAI density changes in the CC and Och were quantified at 8 h, 28 h, 3 days and 7 days after TBI by β-APP and RMO14 immunocytochemistry.
2.
Results
2.1.
Qualitative analysis
In the immunocytochemistry controls, in which primary antibodies were deleted, no immunoreactivity was observed. The sham group showed no β-APP-immunoreactive (IR) axons and RMO14-IR axons. In the injured rats' sections, β-APP-IR and RMO14-IR axonal morphological changes were present in the Och and CC (Figs. 1–3). In the Och, β-APP-IR swollen axons were detected 8 h post-TBI, with some forming retraction balls (Fig. 1A). At 28 h post-TBI, β-APP-IR axons with their stillswollen profiles were observed, but they appeared thinner compared to their previous time point. Additionally, other axon profiles at 28 h were punctuated dense or wavy axons, some capped with terminal bulbs (Fig. 1B). At 3 and 7 days post-TBI, the extent of β-APP-IR axons in the Och decreased and the injury profile was of swollen axons present at the boundary of the Och (close to optic radiations or adjacent nuclei), rather than at its center. In the CC, at 8 and 28 h post-TBI, the profiles of β-APP-IR axons were that of swollen axons with some retraction balls (Fig. 2A). At 3 and 7 days post-TBI, the extent of β-APP-IR axons decreased and the injury profile was that of swollen axons as well as dispersed axons with a punctuated dense appearance. The β-APP-IR axonal density was significantly (p<0.01) less in the CC at 8 h post-TBI than in the Och at the same time point (Figs. 4A, C). In both the Och and CC, the RMO14-IR axons appeared as dense bands and dispersed with a small-punctuated dense appearance at 8 h and 28 h post-TBI (Fig. 2B). At 3 and 7 days post-TBI, the RMO14-IR axonal profiles appeared as elongated dense bands, some capped with terminal bulbs (Fig. 3). The RMO14-IR axonal densities across all time points were statistically insignificant between the Och and the CC. In addition, RMO14-IR axons were mainly present at the boundary of the Och, whereas in the CC the RMO14-IR axons were more diffuse in location.
2.2.
Quantitative analysis
Quantitative analysis of β-APP and RMO14-IR axons over time showed significant changes in the Och. β-APP-IR axons were
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Fig. 1 – Injured axons at 8 h (A) and 28 h (B) post-TBI in the Och demonstrating the morphology of IAT by β-APP-IR. The axonal injury profile at 8 h post-TBI was in the form of swollen axons (white arrow) and retraction balls (black arrow). The axonal injury profile at 28 h post-TBI was in the form of swollen axons (white arrow), punctuated dense appearance (black arrowhead), and wavy dense axons capped with terminal bulbs (white arrowhead). (40× magnification) Scale bar of 100 μm.
most prominent at 8 h post-TBI and significantly decreased with increased survival periods (Fig. 4A). The mean density of β-APP-IR axons in the Och decreased from 109.27±14.98 IR/mm2 at 8 h post-TBI to 29.43±3.36 IR/mm2, 7.56±1.68 IR/mm2, and 3.3± 0.84 IR/mm2 at 28 h, 3 days, and 7 days post-TBI, respectively (Fig. 4A; p<0.01). The mean density of RMO14-IR axons in the Och was most prominent at 28 h post-TBI and decreased thereafter. The mean density of RMO14-IR axons in the Och increased from 7.44 ± 2.70 IR/mm2 at 8 h post-TBI to 20.08 ± 5.05 IR/mm2 at 28 h postTBI, but the change was not statistically significant (Fig. 4B). However, by 3 and 7 days, the mean density of RMO14-IR axons significantly decreased to 3.06± 0.95 IR/mm2 and 6.11 ± 1.11 IR/mm2, respectively (Fig. 4B; p < 0.01). Quantitative analyses of changes in the CC also showed a similar injury profile between β-APP and RMO14-IR axons. The mean density of β-APP-IR axons increased from 26.76 ± 16.66 IR/mm2 at 8 h post-TBI to 72.42 ± 56.11 IR/mm2 at 28 h post-TBI, and then decreased to 28.26± 16.67 IR/mm2 and 12.00 ± 11.01 IR/mm2 at 3 and 7 days post-TBI, respectively (Fig. 4C). The mean density of RMO14-IR axons increased from 14.46 ±
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Fig. 2 – Injured axons at 28 h post-TBI within the CC demonstrating the morphology of IAT by β-APP-IR (A) and NFC by RMO14-IR (B). The profile for β-APP-IR axons was in the form of swollen axons (white arrow) and retraction balls (black arrow), whereas the injury profile for RMO14-IR axons was dense bands (white arrowhead) and small punctuated dense appearance (black arrowhead). (40 × magnification) Scale bar of 100 μm.
6.57 IR/mm2 at 8 h post-TBI to 61.54 ± 43.23 IR/mm2 at 28 h postTBI, and then decreased to 12.44 ± 10.04 IR/mm2 and 26.19 ± 21.45 IR/mm2 at 3 and 7 days post-TBI, respectively (Fig. 4D). However, these changes were not statistically significant, except between β-APP-IR axonal density at 28 h and 7 days post-TBI (Fig. 4C; p <0.05). In the Och and CC, the linear relationship between the density of β-APP and RMO14-IR axons was determined for all rats (n= 24) regardless of their survival time (Fig. 5). In the CC, a strong linear relationship was observed between β-APP-IR and RMO14-IR axons (Fig. 5A; R2 = 0.831, p < 0.01). Because β-APP-IR and RMO14-IR axonal density data points in the CC contained an outlier and many data points near zero, the removal of the outlier and any β-APP-IR and RMO14-IR axonal densities below 3 IR/mm2 further confirmed the strength of the linear relationship. The data points, not including the outlier or any axonal densities below 3 IR/mm2, still showed a significant linear correlation (n = 11, R 2 = 0.401, p < 0.05). However, no linear relationship was observed in the Och between β-APP-IR and RMO14-IR axons (Fig. 5B). Furthermore, the data were subdivided into their respective survival time point groups (8 h, 28 h, 3 days and 7 days; n = 6 per group). In the CC, the linear relationship between
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Fig. 3 – Injured axons at 3 days (A) and 7 days (B) post-TBI within the CC demonstrating the morphology of NFC by RMO14-IR. The axonal injury profile at these time points was dense elongated bands (white arrowhead) and dense bands capped with terminal bulbs (black arrow). (40× magnification) Scale bar of 100 μm.
β-APP-IR and RMO14-IR axons was strong at all survival time points. At 8 h and 3 days post-TBI, the Pearson correlation was 0.872 (p< 0.05; 2 tailed test) between β-APP-IR and RMO14-IR axons. At 28 h and 7 days post-TBI, the Pearson correlation was 0.994 and 0.998 respectively (p < 0.01; 2 tailed test) between β-APP-IR and RMO14-IR axons. For all survival time points, the Och showed no linear relationship between the β-APP-IR and RMO14-IR axons.
3.
Discussion
3.1. Relationship between impaired axonal transport and neurofilament compaction Previous studies have showed that the impact acceleration model induces TAI in the CC (Foda and Marmarou, 1994; Kallakuri et al., 2003; Kallakuri et al., 2012; Li et al., 2011a, 2011b) and the Och (Ding et al., 2001), but only changes that occurred up to 24 h post-TBI were addressed. This study quantified TAI in the CC and Och up to 7 days after TBI in adult rats. Axonal pathology in the CC and Och elicited by immunoreactivity of β-APP and RMO14 revealed distinct morphological
changes. In previous studies, the profiles for β-APP-IR axons were described as swollen and bulbous in shape referred to as retraction balls, whereas the profiles for RMO14-IR axons were described as dense bands or compact linear shapes (Marmarou and Povlishock, 2006; Marmarou et al., 2005; Stone et al., 2001; Suehiro et al., 2001). Similarly, the present study showed βAPP-IR axons consisted of swollen axons and retraction balls, while RMO14-IR axons profiles were in the form of dense bands. Our study also found that at 3 and 7 days post-TBI, the RMO14-IR axonal profiles appeared as elongated dense bands, some capped with terminal bulbs. Our observations revealed that RMO14-IR axons progress to become terminal bulbs over time. In previous studies, the evaluation of NFC in adult rats did not describe the terminal bulb formation of RMO14-IR axons; however, these studies reported TAI in the brainstem and not in the subcortical WM tracts (Marmarou and Povlishock, 2006; Marmarou et al., 2005; Stone et al., 2001; Suehiro et al., 2001). A study in immature rats has reported terminal bulb formation of RMO14-IR axons in the cingulum (DiLeonardi et al., 2009). Immunocytochemistry studies utilizing single or dual labeling of β-APP and RMO14 have showed that NFC and IAT are independent injuries in some axons while dependent in others (DiLeonardi et al., 2009; Marmarou and Povlishock, 2006; Marmarou et al., 2005; Stone et al., 2001; Suehiro et al., 2001). In small caliber axons, focal axonal injury occurs as IAT, whereas in large caliber axons NFC and IAT are co-localized; these types of TAI are distinguished by focal axonal swelling, which progressively increases over time and ultimately leads to secondary axotomy (Stone et al., 2001). Additionally, some axons exhibit NFC in the absence of IAT; the axons of this type of TAI do not present any swellings and very little progressive change occurs in them over time (Stone et al., 2001). Our initial observation of RMO14-IR axons profiles of dense bands followed by elongated dense bands, some capped with terminal bulbs at a later time, supports the existence of two distinct classes of TAI as reported by Stone et al. in 2001. The RMO14-IR axons that exhibited elongated dense band profiles at 3 and 7 days may belong to the TAI class of injured axons exhibiting NFC alone; whereas, RMO14-IR axons that exhibit elongated dense bands capped with terminal bulbs may belong to the TAI class of injured axons exhibiting co-localized NFC and IAT. In this study, the Och showed no linear relationship between the density of β-APP-IR axons and RMO14-IR axons, suggesting that IAT and NFC occur in different axonal populations in the Och. However, the CC showed a strong linear correlation between β-APP-IR axons and RMO14-IR axons consistent with DiLeonardi et al. (2009), who reported that in the CC, β-APP and RMO14-IR axons occurred in the same anatomical location and at 3 days post-TBI, while β-APP and RMO14-IR axons were intermingled but occurred in independent axons. In addition, DiLeornardi et al. reported that other structures such as the cingulum did not have co-localized β-APP and RMO14-IR axons. The present study similarly showed the complexity of TAI progression and its dependence on the WM structure.
3.2.
Temporal profile of traumatic axonal injury
In this study, peak β-APP-IR density occurred at 8 h in the Och and 28 h in the CC. In rats, the Och is composed of large
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Fig. 4 – Representation of the quantified mean density of β-APP-IR axons (A, C) and RMO14-IR (B, D) axons in Och (A, B) and CC (C, D) at 8 h, 28 h, 3 days and 7 days post-injury. Within the Och, β-APP-IR axons were most prominent at 8 h post-TBI and significantly decreased as time elapsed (A). RMO14-IR axons within the Och were most prominent at 28 h post-TBI and significantly decreased as time elapsed (B). Within the CC (C, D), β-APP and RMO14-IR axons were more prominent at 28 h. Differences of TAI densities between each time point within the CC were not significant except between β-APP-IR axons at 28 h and 7 days post-TBI (C, D). Values represent mean density and the error bars represent standard error of the mean.
myelinated axons (Duvdevani et al., 1993), and this structure displayed its greatest β-APP-IR density at an early point in time post impact. This finding agrees with trends in other studies using the Marmarou model, where large caliber axons of the thoracolumbar region in the spinal cord were found to display their greatest density of positive β-APP at 6 h postinjury, which is earlier than described for brainstem tracts (Czeiter et al., 2008; Marmarou et al., 2005). The CC is composed of a heterogeneous population of unmyelinated (diameter: 0.1–0.7 μm) and myelinated (diameter: 0.3–1.0 μm) axons (Waxman and Black, 1988). This composition may possibly contribute to the wide variation of the CC injury density observed in the present study. Additionally, the CC axons (Waxman and Black, 1988), which are of smaller caliber than Och axons (Duvdevani et al., 1993), exhibited the greatest β-APP-IR density at 28 h post impact. Smaller-caliber axons possess proportionally greater numbers of microtubules per unit area (Friede and Samorajski, 1970; Malbouisson et al., 1985), and larger-caliber axons possess proportionally greater numbers of neurofilaments per unit area (Price et al., 1988). Additionally, an electrophysiology study of compound action potential (CAP) of injured axons in the CC reported the greatest decrease of CAP from fast conducting myelinated fibers
(N1) and slow conducting unmyelinated fibers (N2) at 24 h postTBI (Reeves et al., 2005). Similarly, in our study both β-APP-IR and RMO14-IR axonal densities peaked within the CC at 28 h post-TBI. Also, Reeves et al. (2005) reported that N1 wave amplitude increased at 3 days post-TBI and further increased to a significant recovery by 7 days post-TBI, with no recovery of N2 amplitude. In our study, IR axons in the CC remained present at 7 days post-TBI, suggesting a relation to N2 unmyelinated fiber changes. The quantitative TAI changes in the CC did not show significant differences over time, except between β-APP-IR axonal density at 28 h and 7 days post-TBI; this may be related to the wide variation of the CC injury density among the rats, which has been observed previously (Li et al., 2011a). One study reported that in the ventral brainstem, as time elapsed from 5 min to 60 min, NFC increased and became most prominent at 24 h post-TBI (Povlishock et al., 1997). This was also the case in the present study where RMO14-IR increased from 8 h post-TBI and became most prominent at 28 h post-TBI.
3.3.
Clinical relevance
In this study the peak expression of TAI differed in time for the CC and Och. Also, NFC and IAT presented varying injury
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Fig. 5 – Correlation of RMO14-IR axonal density with β-APP-IR axonal density in the CC (A) and Och (B) for all survival time points. Each circle represents one rat. A strong positive linear correlation was observed between RMO14-IR axonal density and β-APP-IR axonal density in the CC (A) (R2 = 0.831; p < 0.001). Dashed lines represent 95% mean confidence intervals.
density time profiles. These findings suggest that a therapeutic time window is of importance in addressing different pathologies, as it has previously been reported (Loane and Faden, 2010; Xiong et al., 2009). The current findings in CC and Och suggest that the therapeutic time window may vary depending on the WM structures that are affected. Various studies have showed that multiple immunocytochemical approaches are needed to assess the full spectrum of TAI (DiLeonardi et al., 2009; Marmarou et al., 2005; Stone et al., 2001). Initially the accumulation of β-APP occurs in the absence of axolemmal permeability; however, the transport impairment may lead to delayed membrane damage and axolemma permeability (Stone et al., 2004). Treatment targeting IAT may be crucial at earlier time points, as suggested by its peak at 8 h in the Och and 28 h in the CC in this study. Additionally,
horseradish peroxidase investigations have illustrated that axons experiencing axolemma permeability changes will tend to exhibit NFC (Okonkwo et al., 1998; Pettus and Povlishock, 1996; Pettus et al., 1994; Povlishock et al., 1997, 1999). When the axolemmal permeability is altered as a result of TBI, Ca2+ influx and intracellular Ca2+ concentration will increase (Giza and Hovda, 2001; Marmarou et al., 2005; Maxwell and Graham, 1997; Povlishock, 1992; Smith et al., 2003). The increased Ca2+ activates calcineurin (calcium-activated phosphatase) (Hoffman, 1995; Marmarou et al., 2005), which dephosphorylates the neurofilament sidearms, causing NFC. To prevent NFC, various mechanisms can be utilized as treatment. One option is to inhibit the activation of calcineurin by Ca2+, another is to inhibit the dephosphorylation of neurofilament sidearms by calcineurin (Buki et al., 1999b, 2003; Suehiro et al., 2001).
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Our study also suggests that subcellular aspects of TAI occur dependently and/or independently of each other, thereby supporting the use of a multi-drug therapeutic approach to target the full spectrum of TAI.
3.4.
Conclusions
The TAI density was widely variable in the CC and consistent in the Och among the rats. Varying temporal profiles of IAT and NFC were observed in both structures. The density of β-APP-IR axons peaked at 8 h in the CC and 28 h in the Och, whereas the density of RMO14-IR axons peaked at 28 h in both the CC and the Och. After peaking, TAI density decreased over time and remained detectable at 7 days. Additionally, a strong positive linear relationship (R2 = 0.831) between β-APP-IR and RMO14-IR axonal density was observed in the CC but not in the Och. It is suggested that multiple markers are required to fully evaluate TAI. Because each subcellular aspect of TAI demonstrates different profiles over time, utility of diverse pharmacological agents tailored to target various pathological events occurring at different time points may be useful.
4.
Experimental procedure
The institutional animal care and use committee approved all procedures in this study. Twenty-six adult male Sprague Daley rats weighing 394 ± 39.6 g (mean ± SD) (Harlan, IN) were utilized. Twenty-four rats were randomly assigned to four different post-TBI survival time points (n= 6/time point) and two rats were assigned as sham rats. The sham rats were not subjected to surgery and TBI induction. All rats had free access to food and water.
prior to impact and 4 h, 24 h, 3 days and 7 days post impact), each rat was initially anesthetized in a sealed acrylic chamber by a mixture of 2% isoflurane and 0.6 L/min oxygen and maintained under anesthesia for 4 h during the imaging procedure by a mixture of 0.75% to 1.75% isoflurane and 0.6 L/min of oxygen via nose cone. Because the MRI acquisition length was 4 h, the first histological time points in this study were 8 h and 28 h postTBI. At the end of designated survival time (8 h, 28 h, 3 days and 7 days post-TBI), the injured rats were euthanized with an overdose of sodium pentobarbital and exsanguinated. The rats were then transcardially perfused with normal saline followed by cold 4% paraformaldehyde in phosphate buffered saline (0.1 M, pH 7.45). Following perfusion, the brains were carefully removed and post fixed in 4% paraformaldehyde with 20% sucrose. The post fixed brain was cut into a block encompassing the genu and the splenium of the CC which was embedded in Tissue-Tek® O.C.T compound and frozen at −78.5 °C via dry ice. A series of 40 μm coronal frozen sections were then cut from the block (Leica CM 3050, Leica Microsystems GmbH, Heidelberg Germany) and collected in multi-well plates. To represent the Och, two consecutive sections were selected from the anterior, middle and posterior regions of the Och ranging in distance from 0.13 to 0.84 mm posterior to bregma. To represent the CC, three sets of two consecutive sections of 280 μm apart were selected from the caudal portion of the CC. The sections selected to represent TAI in the caudal of the CC were approximately 3–4.5 mm posterior to bregma. Therefore, six sections of the CC and six sections of the Och were processed for β-APP (CC n = 3, Och n = 3), and RMO14 (CC n = 3, Och n = 3) immunocytochemistry analysis.
4.2. 4.1.
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Immunochemistry for IAT and NFC
Surgical preparation and TBI induction
TBI was induced by Marmarou impact acceleration head injury model (Foda and Marmarou, 1994; Marmarou et al., 1994). Initially all rats were anesthetized by 2% to 2.5% isoflurane and 0.6 L/min oxygen in an anesthesia chamber. To induce TBI after initial anesthesia, the rats were maintained with 1% to 1.75% isoflurane and 0.6 L/min oxygen via a nose cone. Then a midline incision was preformed to expose the periosteum covering the vertex of the skull (Marmarou et al., 1994). The periosteum was reflected and a 10 mm diameter steel disk with a thickness of 3 mm affixed by cranioplastic powder (Plastic One, Roanoke, VA) at the midline between the bregma and lamboid sutures. The rats were positioned in a prone position on a foam bed (12×12×43 cm) contained in a Plexiglas box and taped in place around their trunk in order to prevent them from falling off the foam bed after trauma induction. Anesthesia was removed just prior to TBI induction. TBI was induced by dropping a 450 g cylindrical brass weight of 18 mm in diameter from a height of 2 m onto the vertex of the skull at the center of the disk. After the impact, the disk was carefully removed and the skull examined for fractures. Rats with no skull fractures had their skin sutured and were allowed to recover. This study is a part of a parallel imaging study and each rat was maintained under anesthesia for 4 h during the imaging procedure. At the designated imaging time points (at least 1 day
Of the two consecutive sections selected, one section was utilized to stain for β-APP and the other for RMO14. The sections were rinsed 3 × 3 min in phosphate-buffered saline (PBS) and then processed for antigen retrieval by incubation in citrate buffer for 1 h at 75–80 °C. The sections were allowed to cool in the same citrate buffer solution for 30 min and then rinsed 3 × 3 min in PBS. Subsequently, to quench endogenous peroxidase activity the sections were incubated in 0.3% hydrogen peroxidase for 1 h and then rinsed 3 × 3 min in PBS. The brain sections were then incubated overnight either in C-terminus specific APP primary antibody (1 μg/ml; rabbit anti-C-terminus β-APP; cat # 51-2700; Zymed, San Francisco, CA) or in RMO14 primary antibody (1 μg/ml; mouse anti-NF-M; cat # 34-1000; Zymed, San Francisco, CA) with 0.01% Triton X and 1% albumin bovine serum in 2% normal goat serum and PBS. The sections were rinsed 3× 3 min in PBS. The sections that were incubated in APP primary antibody and RMO14 primary antibody were incubated for 2 h in goat anti-rabbit IgG or goat anti-mouse IgG secondary antibody (Vector Laboratories, Burlingame, CA) respectively. The sections were rinsed 3× 3 min in PBS and incubated for 1.5 h in avidin biotin peroxidase complex (Vectastain ABC Standard Elite Kit, Vector). The sections were rinsed 3× 3 min in PBS and briefly incubated in 3, 3′-diaminobenzidine and hydrogen peroxide. Lastly, the brain sections were rinsed 3× 5 min in PBS, dehydrated and cover-slipped using Permount.
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Negative control incubations were performed in the absence of primary antibody.
4.3.
Digital image acquisition
Serial photomicrographs for each coronal section (20× magnification, height= 436.48 μm; length = 327.04 μm; 3.18 pixels/μm) were obtained to encompass the whole region of interest (i.e., Och or CC) by a Zeiss Axio Observer Inverted Microscope (Carl Zeiss, Inc.) fitted with Axio MRC digital camera. The digital images were exported into Adobe Photoshop CS2 for construction of panoramic images of the region of interest (Figs. 6A, D) by photomerging the serial photomicrographs. The saved JPEG panoramic images were imported into ImageJ (http://rsb.info. nih.gov/ij/) to quantify the density of β-APP-IR and RMO14-IR axons.
4.4.
TAI quantification
The panoramic images were viewed in ImageJ on a computer monitor to clearly delineate the Och and CC (Figs. 6B, E). For the Och, the boundaries and the surrounding tissue were digitally removed (Fig. 6C). For the CC, the delineated area was centered on the midline (Fig. 6E) and the tissue outside the delineated area removed (Fig. 6F). The surface area of each delineated region was computed by ImageJ software. These delineated areas of CC and Och were further amplified on the monitor via ImageJ software. Within the delineated areas, the TAI was
quantified by clicking the mouse on the β-APP-IR or RMO14-IR axons, and the cell counter feature in ImageJ software computed the total immunopositive axonal count. β-APP-IR axons that appeared as dense immunopositive swollen profiles and as retraction balls were quantified as injured axons. RMO14-IR axons that appeared as dense immunopositive bands or had a dense punctuated appearance were quantified as injured axons. The injured axonal density was then computed by dividing the sum of the total number of β-APP-IR or RMO14-IR in each panoramic delineated region of interest by the sum of the corresponding delineated areas in square millimeters.
4.5.
Statistical methods
All data were analyzed using SPSS version 17 for Windows. The numbers of injured axons were expressed as the mean value ± standard error of the mean (SEM). The data distribution was tested using the Shapiro–Wilk normality test (p ≤0.05) and Levene's test of homogeneity of variance (p ≤0.05). The mean values of the data set were either not normally distributed as indicated by the Shapiro–Wilk test, or the variances of the data set were heterogeneous as indicated by Levene's test. As the Kruskal–Wallis test does not assume normality in the data set and is insensitive to outliers in the data, this test was utilized to compare the four time points. If the Kruskal–Wallis test showed significance (p≤0.05), then the Mann–Whitney exact test was utilized for pairwise comparisons between two time points. Statistical significance was set at a p value of less than 0.05.
Fig. 6 – Panoramic images of the Och (A) and CC (D). The black outline in Och (B) and CC (E) shows the boundary of the anatomical structures analyzed. Panels C and F represent the area within the outline of the anatomical structure that was analyzed. (20 × magnification) Scale bar of 250 μm.
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Acknowledgments The authors would like to thank Dr. Bulent Ozkan for his advice and guidance on the statistical analysis. The valuable help of Mr. Branden Anderson and Mr. Gurjiwan Virk for data collection is also highly appreciated. Special thanks to Mrs. Charlotte Harman and to Ms. Beth Langelier for editorial assistance.
REFERENCES
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neurofilament compaction occurring with traumatic axonal injury. Brain Res. 784, 1–6. Pal, J., Toth, Z., Farkas, O., Kellenyi, L., Doczi, T., Gallyas, F., 2006. Selective induction of ultrastructural (neurofilament) compaction in axons by means of a new head-injury apparatus. J. Neurosci. Methods 153, 283–289. Perunovic, B., Quilty, R.D., Athanasiou, A., Love, S., 2001. Damage to intracranial optic pathways in fatal closed head injury in man. J. Neurol. Sci. 185, 55–62. Pettus, E.H., Povlishock, J.T., 1996. Characterization of a distinct set of intra-axonal ultrastructural changes associated with traumatically induced alteration in axolemmal permeability. Brain Res. 722, 1–11. Pettus, E.H., Christman, C.W., Giebel, M.L., Povlishock, J.T., 1994. Traumatically induced altered membrane permeability: its relationship to traumatically induced reactive axonal change. J. Neurotrauma 11, 507–522. Povlishock, J.T., 1992. Traumatically induced axonal injury: pathogenesis and pathobiological implications. Brain Pathol. 2, 1–12. Povlishock, J.T., Christman, C.W., 1995. The pathobiology of traumatically induced axonal injury in animals and humans: a review of current thoughts. J. Neurotrauma 12, 555–564. Povlishock, J.T., Marmarou, A., McIntosh, T., Trojanowski, J.Q., Moroi, J., 1997. Impact acceleration injury in the rat: evidence for focal axolemmal change and related neurofilament sidearm alteration. J. Neuropathol. Exp. Neurol. 56, 347–359. Povlishock, J.T., Buki, A., Koiziumi, H., Stone, J., Okonkwo, D.O., 1999. Initiating mechanisms involved in the pathobiology of traumatically induced axonal injury and interventions targeted at blunting their progression. Acta Neurochir. Suppl. 73, 15–20. Price, R.L., Paggi, P., Lasek, R.J., Katz, M.J., 1988. Neurofilaments are spaced randomly in the radial dimension of axons. J. Neurocytol. 17, 55–62.
Reeves, T.M., Phillips, L.L., Povlishock, J.T., 2005. Myelinated and unmyelinated axons of the corpus callosum differ in vulnerability and functional recovery following traumatic brain injury. Exp. Neurol. 196, 126–137. Slazinski, T., Johnson, M.C., 1994. Severe diffuse axonal injury in adults and children. J. Neurosci. Nurs. 26, 151–154. Smith, D.H., Meaney, D.F., Shull, W.H., 2003. Diffuse axonal injury in head trauma. J. Head Trauma Rehabil. 18, 307–316. Stone, J.R., Singleton, R.H., Povlishock, J.T., 2000. Antibodies to the C-terminus of the beta-amyloid precursor protein (APP): a site specific marker for the detection of traumatic axonal injury. Brain Res. 871, 288–302. Stone, J.R., Singleton, R.H., Povlishock, J.T., 2001. Intra-axonal neurofilament compaction does not evoke local axonal swelling in all traumatically injured axons. Exp. Neurol. 172, 320–331. Stone, J.R., Okonkwo, D.O., Dialo, A.O., Rubin, D.G., Mutlu, L.K., Povlishock, J.T., Helm, G.A., 2004. Impaired axonal transport and altered axolemmal permeability occur in distinct populations of damaged axons following traumatic brain injury. Exp. Neurol. 190, 59–69. Suehiro, E., Singleton, R.H., Stone, J.R., Povlishock, J.T., 2001. The immunophilin ligand FK506 attenuates the axonal damage associated with rapid rewarming following posttraumatic hypothermia. Exp. Neurol. 172, 199–210. Trojanowski, J.Q., Kelsten, M.L., Lee, V.M., 1989. Phosphate-dependent and independent neurofilament protein epitopes are expressed throughout the cell cycle in human medulloblastoma (D283 MED) cells. Am. J. Pathol. 135, 747–758. Waxman, S.G., Black, J.A., 1988. Unmyelinated and myelinated axon membrane from rat corpus callosum: differences in macromolecular structure. Brain Res. 453, 337–343. Xiong, Y., Mahmood, A., Chopp, M., 2009. Emerging treatments for traumatic brain injury. Expert Opin. Emerg. Drugs 14, 67–84.
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Research Report
Temporal assessment of traumatic axonal injury in the rat corpus callosum and optic chiasm Nisrine Zakaria⁎, Srinivasu Kallakuri1 , Sharath Bandaru1 , John M. Cavanaugh1 College of Engineering, Department of Biomedical Engineering, Wayne State University, Detroit, MI 48202, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
Impaired axoplasmic transport (IAT) and neurofilament compaction (NFC), two common
Accepted 22 May 2012
axonal pathology processes involved in traumatic axonal injury (TAI), have been well
Available online 28 May 2012
characterized. TAI is found clinically and in animal models in brainstem white matter (WM) tracts and in the corpus callosum (CC), optic chiasm (Och), and internal capsule. Previous
Keywords:
published quantitative studies of the time course of TAI expression induced by the
Traumatic axonal injury
Marmarou impact acceleration model have been limited to the brainstem. Accordingly, this
Corpus callosum
study assessed the extent of IAT and NFC in the CC and Och at 8 h, 28 h, 3 days and 7 days
Optic chiasm
after traumatic brain injury (TBI) induction by the Marmarou impact acceleration model.
Impaired axoplasmic transport
IAT peak density was observed at 8 h in the CC and 28 h in the Och post-TBI. NFC peak
Neurofilament compaction
density was observed at 28 h in both structures. The density of IAT and NFC decreased with
Impact acceleration model
increasing survival time in both structures. The NFC density time profile followed a similar trend in both the Och and CC, whereas the IAT density time profile was variable between the Och and CC. Furthermore, a strong linear relationship was observed between IAT and NFC in the CC but not in the Och. These findings highlight the heterogeneity of TAI as evidenced by variable IAT and NFC injury time profiles in each anatomical structure. This variability indicates the requirement of multiple markers for a comprehensive TAI evaluation and multiple targeted treatments for TAI polypathology within its therapeutic window time frame. © 2012 Elsevier B.V. All rights reserved.
1.
Introduction
Diffuse axonal injury (DAI), also referred to as traumatic axonal injury (TAI), is a well-recognized consequence of blunt head
injury (Adams et al., 1982). TAI is considered a major contributor to morbidity and mortality after traumatic brain injury (TBI) (Adams et al., 1989; Bennett et al., 1995; Gentleman et al., 1995; Povlishock and Christman, 1995; Slazinski and Johnson, 1994).
⁎ Corresponding author at: Department of Biomedical Engineering, 818 West Hancock, Wayne State University, Detroit, MI 48201, USA. Fax: + 1 313 577 8333. E-mail addresses: [email protected] (N. Zakaria), [email protected] (S. Kallakuri), [email protected] (S. Bandaru), [email protected] (J.M. Cavanaugh). Abbreviations: β-APP, beta amyloid precursor protein; CC, corpus callosum; DAI, diffuse axonal injury; IAT, impaired axoplasmic transport; IR, immunoreactive; NFC, neurofilament compaction; NF-M, neurofilament medium; Och, optic chiasm; TAI, traumatic axonal injury; TBI, traumatic brain injury; WM, white matter 1 Fax: +1 313 577 8333. 0006-8993/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2012.05.046
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TAI is produced by rapid head acceleration/deceleration during a traumatic event (Kelley et al., 2006) with consequent tension or shear on axons. Clinically, TAI has been reported to appear throughout the deep and subcortical white matter (WM) and has been noted to be predominantly common in the midline structures, including the corpus callosum (CC) and brainstem (Meythaler et al., 2001; Smith et al., 2003). In addition, vision impairment has also been reported to occur clinically following closed head injury (Perunovic et al., 2001). A post-mortem immunocytochemistry study of severely closed head injury cases revealed the presence of TAI at different sites within the optic pathways, such as the optic chiasm (Och), optic tract and optic radiations (Perunovic et al., 2001). The Marmarou impact acceleration weight-drop model has been widely used since its origination in 1994 to induce closed head TBI in rats in order to study TAI and other pathological changes. This model has been reported to induce DAI, particularly in the CC, internal capsule, optic tracts, cerebral and cerebellar peduncles and in the long tracts in the brainstem (Foda and Marmarou, 1994). Despite clinical findings of TAI in the subcortical WM and optic pathways, laboratory studies of TAI in these anatomical structures are limited, especially those using impact acceleration injury models. The majority of TAI studies using the Marmarou model have focused on the brainstem (Buki et al., 2000; Marmarou and Povlishock, 2006; Marmarou et al., 2005; Povlishock et al., 1997; Stone et al., 2000, 2001, 2004; Suehiro et al., 2001). The original histological study of the impact acceleration model by Foda and Marmarou (1994) reported that TAI does occur in the brainstem and to a lesser extent in the cortical WM. To the best of our knowledge, only five histological studies utilizing the Marmarou model have reported TAI in the cortical WM in a quantitative manner (Ding et al., 2001; Kallakuri et al., 2003; Kallakuri et al., 2012; Li et al., 2011a; Li et al., 2011b). These studies showed that it is possible to induce TAI in the CC (Kallakuri et al., 2003; Kallakuri et al., 2012; Li et al., 2011a, 2011b) and optic chiasm (Och) (Ding et al., 2001) using the Marmarou model. Because clinical studies have indicated that the CC and Och pathways are prominent areas of TAI after TBI, further laboratory studies are needed to assess the extent of TAI in these regions following closed-head TBI. The few laboratory studies that have quantified TAI in the CC (Kallakuri et al., 2003; Kallakuri et al., 2012; Li et al., 2011a, 2011b) and the Och (Ding et al., 2001) using this model are limited to studying TAI expression at 24 h post-TBI. However, a temporal assessment is lacking of TAI density in these structures following TBI induced by impact acceleration. Beta amyloid precursor protein (β-APP) immunostaining has been found valuable in the detection of TAI as early as 30–35 min after the traumatic event in rats and humans (Gorrie et al., 2002; Hortobagyi et al., 2007; Marmarou et al., 2005; Stone et al., 2001) and was used extensively as a marker of impaired axoplasmic transport (IAT) (DiLeonardi et al., 2009; Geddes et al., 2003; Gorrie et al., 2002; Hortobagyi et al., 2007; Marmarou et al., 2005; McKenzie et al., 1996; Pal et al., 2006; Stone et al., 2001; Suehiro et al., 2001). In addition to IAT, the intra-axonal cytoskeletal alteration characterized as neurofilament compaction (NFC) has been increasingly investigated by utilizing specific neurofilament medium chain (NF-M) antibodies (Czeiter et al., 2008; Stone et al., 2001; Suehiro et al., 2001). NFC was shown to be associated with either dephosphorylation or calpain-mediated
proteolysis of neurofilament sidearms (Buki et al., 1999a; Giza and Hovda, 2001; Marmarou et al., 2005). The monoclonal RMO14 antibodies specifically recognize epitopes on the NF-M rod domain that become available after dephosphorylation, enabling the detection of NFC (Trojanowski et al., 1989). IAT and NFC are two distinct processes occurring in distinct axonal populations (Stone et al., 2001). Compared to other histological methods such as silver staining, immunohistochemistry has the advantage of detecting subcellular aspects of TAI such as IAT and NFC (DiLeonardi et al., 2009; Marmarou et al., 2005; Pal et al., 2006; Stone et al., 2001; Suehiro et al., 2001). The purpose of this study was to assess the subcellular aspects of TAI density over time in two clinically relevant WM tracts. Specifically, the temporal subcellular TAI density changes in the CC and Och were quantified at 8 h, 28 h, 3 days and 7 days after TBI by β-APP and RMO14 immunocytochemistry.
2.
Results
2.1.
Qualitative analysis
In the immunocytochemistry controls, in which primary antibodies were deleted, no immunoreactivity was observed. The sham group showed no β-APP-immunoreactive (IR) axons and RMO14-IR axons. In the injured rats' sections, β-APP-IR and RMO14-IR axonal morphological changes were present in the Och and CC (Figs. 1–3). In the Och, β-APP-IR swollen axons were detected 8 h post-TBI, with some forming retraction balls (Fig. 1A). At 28 h post-TBI, β-APP-IR axons with their stillswollen profiles were observed, but they appeared thinner compared to their previous time point. Additionally, other axon profiles at 28 h were punctuated dense or wavy axons, some capped with terminal bulbs (Fig. 1B). At 3 and 7 days post-TBI, the extent of β-APP-IR axons in the Och decreased and the injury profile was of swollen axons present at the boundary of the Och (close to optic radiations or adjacent nuclei), rather than at its center. In the CC, at 8 and 28 h post-TBI, the profiles of β-APP-IR axons were that of swollen axons with some retraction balls (Fig. 2A). At 3 and 7 days post-TBI, the extent of β-APP-IR axons decreased and the injury profile was that of swollen axons as well as dispersed axons with a punctuated dense appearance. The β-APP-IR axonal density was significantly (p<0.01) less in the CC at 8 h post-TBI than in the Och at the same time point (Figs. 4A, C). In both the Och and CC, the RMO14-IR axons appeared as dense bands and dispersed with a small-punctuated dense appearance at 8 h and 28 h post-TBI (Fig. 2B). At 3 and 7 days post-TBI, the RMO14-IR axonal profiles appeared as elongated dense bands, some capped with terminal bulbs (Fig. 3). The RMO14-IR axonal densities across all time points were statistically insignificant between the Och and the CC. In addition, RMO14-IR axons were mainly present at the boundary of the Och, whereas in the CC the RMO14-IR axons were more diffuse in location.
2.2.
Quantitative analysis
Quantitative analysis of β-APP and RMO14-IR axons over time showed significant changes in the Och. β-APP-IR axons were
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Fig. 1 – Injured axons at 8 h (A) and 28 h (B) post-TBI in the Och demonstrating the morphology of IAT by β-APP-IR. The axonal injury profile at 8 h post-TBI was in the form of swollen axons (white arrow) and retraction balls (black arrow). The axonal injury profile at 28 h post-TBI was in the form of swollen axons (white arrow), punctuated dense appearance (black arrowhead), and wavy dense axons capped with terminal bulbs (white arrowhead). (40× magnification) Scale bar of 100 μm.
most prominent at 8 h post-TBI and significantly decreased with increased survival periods (Fig. 4A). The mean density of β-APP-IR axons in the Och decreased from 109.27±14.98 IR/mm2 at 8 h post-TBI to 29.43±3.36 IR/mm2, 7.56±1.68 IR/mm2, and 3.3± 0.84 IR/mm2 at 28 h, 3 days, and 7 days post-TBI, respectively (Fig. 4A; p<0.01). The mean density of RMO14-IR axons in the Och was most prominent at 28 h post-TBI and decreased thereafter. The mean density of RMO14-IR axons in the Och increased from 7.44 ± 2.70 IR/mm2 at 8 h post-TBI to 20.08 ± 5.05 IR/mm2 at 28 h postTBI, but the change was not statistically significant (Fig. 4B). However, by 3 and 7 days, the mean density of RMO14-IR axons significantly decreased to 3.06± 0.95 IR/mm2 and 6.11 ± 1.11 IR/mm2, respectively (Fig. 4B; p < 0.01). Quantitative analyses of changes in the CC also showed a similar injury profile between β-APP and RMO14-IR axons. The mean density of β-APP-IR axons increased from 26.76 ± 16.66 IR/mm2 at 8 h post-TBI to 72.42 ± 56.11 IR/mm2 at 28 h post-TBI, and then decreased to 28.26± 16.67 IR/mm2 and 12.00 ± 11.01 IR/mm2 at 3 and 7 days post-TBI, respectively (Fig. 4C). The mean density of RMO14-IR axons increased from 14.46 ±
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Fig. 2 – Injured axons at 28 h post-TBI within the CC demonstrating the morphology of IAT by β-APP-IR (A) and NFC by RMO14-IR (B). The profile for β-APP-IR axons was in the form of swollen axons (white arrow) and retraction balls (black arrow), whereas the injury profile for RMO14-IR axons was dense bands (white arrowhead) and small punctuated dense appearance (black arrowhead). (40 × magnification) Scale bar of 100 μm.
6.57 IR/mm2 at 8 h post-TBI to 61.54 ± 43.23 IR/mm2 at 28 h postTBI, and then decreased to 12.44 ± 10.04 IR/mm2 and 26.19 ± 21.45 IR/mm2 at 3 and 7 days post-TBI, respectively (Fig. 4D). However, these changes were not statistically significant, except between β-APP-IR axonal density at 28 h and 7 days post-TBI (Fig. 4C; p <0.05). In the Och and CC, the linear relationship between the density of β-APP and RMO14-IR axons was determined for all rats (n= 24) regardless of their survival time (Fig. 5). In the CC, a strong linear relationship was observed between β-APP-IR and RMO14-IR axons (Fig. 5A; R2 = 0.831, p < 0.01). Because β-APP-IR and RMO14-IR axonal density data points in the CC contained an outlier and many data points near zero, the removal of the outlier and any β-APP-IR and RMO14-IR axonal densities below 3 IR/mm2 further confirmed the strength of the linear relationship. The data points, not including the outlier or any axonal densities below 3 IR/mm2, still showed a significant linear correlation (n = 11, R 2 = 0.401, p < 0.05). However, no linear relationship was observed in the Och between β-APP-IR and RMO14-IR axons (Fig. 5B). Furthermore, the data were subdivided into their respective survival time point groups (8 h, 28 h, 3 days and 7 days; n = 6 per group). In the CC, the linear relationship between
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Fig. 3 – Injured axons at 3 days (A) and 7 days (B) post-TBI within the CC demonstrating the morphology of NFC by RMO14-IR. The axonal injury profile at these time points was dense elongated bands (white arrowhead) and dense bands capped with terminal bulbs (black arrow). (40× magnification) Scale bar of 100 μm.
β-APP-IR and RMO14-IR axons was strong at all survival time points. At 8 h and 3 days post-TBI, the Pearson correlation was 0.872 (p< 0.05; 2 tailed test) between β-APP-IR and RMO14-IR axons. At 28 h and 7 days post-TBI, the Pearson correlation was 0.994 and 0.998 respectively (p < 0.01; 2 tailed test) between β-APP-IR and RMO14-IR axons. For all survival time points, the Och showed no linear relationship between the β-APP-IR and RMO14-IR axons.
3.
Discussion
3.1. Relationship between impaired axonal transport and neurofilament compaction Previous studies have showed that the impact acceleration model induces TAI in the CC (Foda and Marmarou, 1994; Kallakuri et al., 2003; Kallakuri et al., 2012; Li et al., 2011a, 2011b) and the Och (Ding et al., 2001), but only changes that occurred up to 24 h post-TBI were addressed. This study quantified TAI in the CC and Och up to 7 days after TBI in adult rats. Axonal pathology in the CC and Och elicited by immunoreactivity of β-APP and RMO14 revealed distinct morphological
changes. In previous studies, the profiles for β-APP-IR axons were described as swollen and bulbous in shape referred to as retraction balls, whereas the profiles for RMO14-IR axons were described as dense bands or compact linear shapes (Marmarou and Povlishock, 2006; Marmarou et al., 2005; Stone et al., 2001; Suehiro et al., 2001). Similarly, the present study showed βAPP-IR axons consisted of swollen axons and retraction balls, while RMO14-IR axons profiles were in the form of dense bands. Our study also found that at 3 and 7 days post-TBI, the RMO14-IR axonal profiles appeared as elongated dense bands, some capped with terminal bulbs. Our observations revealed that RMO14-IR axons progress to become terminal bulbs over time. In previous studies, the evaluation of NFC in adult rats did not describe the terminal bulb formation of RMO14-IR axons; however, these studies reported TAI in the brainstem and not in the subcortical WM tracts (Marmarou and Povlishock, 2006; Marmarou et al., 2005; Stone et al., 2001; Suehiro et al., 2001). A study in immature rats has reported terminal bulb formation of RMO14-IR axons in the cingulum (DiLeonardi et al., 2009). Immunocytochemistry studies utilizing single or dual labeling of β-APP and RMO14 have showed that NFC and IAT are independent injuries in some axons while dependent in others (DiLeonardi et al., 2009; Marmarou and Povlishock, 2006; Marmarou et al., 2005; Stone et al., 2001; Suehiro et al., 2001). In small caliber axons, focal axonal injury occurs as IAT, whereas in large caliber axons NFC and IAT are co-localized; these types of TAI are distinguished by focal axonal swelling, which progressively increases over time and ultimately leads to secondary axotomy (Stone et al., 2001). Additionally, some axons exhibit NFC in the absence of IAT; the axons of this type of TAI do not present any swellings and very little progressive change occurs in them over time (Stone et al., 2001). Our initial observation of RMO14-IR axons profiles of dense bands followed by elongated dense bands, some capped with terminal bulbs at a later time, supports the existence of two distinct classes of TAI as reported by Stone et al. in 2001. The RMO14-IR axons that exhibited elongated dense band profiles at 3 and 7 days may belong to the TAI class of injured axons exhibiting NFC alone; whereas, RMO14-IR axons that exhibit elongated dense bands capped with terminal bulbs may belong to the TAI class of injured axons exhibiting co-localized NFC and IAT. In this study, the Och showed no linear relationship between the density of β-APP-IR axons and RMO14-IR axons, suggesting that IAT and NFC occur in different axonal populations in the Och. However, the CC showed a strong linear correlation between β-APP-IR axons and RMO14-IR axons consistent with DiLeonardi et al. (2009), who reported that in the CC, β-APP and RMO14-IR axons occurred in the same anatomical location and at 3 days post-TBI, while β-APP and RMO14-IR axons were intermingled but occurred in independent axons. In addition, DiLeornardi et al. reported that other structures such as the cingulum did not have co-localized β-APP and RMO14-IR axons. The present study similarly showed the complexity of TAI progression and its dependence on the WM structure.
3.2.
Temporal profile of traumatic axonal injury
In this study, peak β-APP-IR density occurred at 8 h in the Och and 28 h in the CC. In rats, the Och is composed of large
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Fig. 4 – Representation of the quantified mean density of β-APP-IR axons (A, C) and RMO14-IR (B, D) axons in Och (A, B) and CC (C, D) at 8 h, 28 h, 3 days and 7 days post-injury. Within the Och, β-APP-IR axons were most prominent at 8 h post-TBI and significantly decreased as time elapsed (A). RMO14-IR axons within the Och were most prominent at 28 h post-TBI and significantly decreased as time elapsed (B). Within the CC (C, D), β-APP and RMO14-IR axons were more prominent at 28 h. Differences of TAI densities between each time point within the CC were not significant except between β-APP-IR axons at 28 h and 7 days post-TBI (C, D). Values represent mean density and the error bars represent standard error of the mean.
myelinated axons (Duvdevani et al., 1993), and this structure displayed its greatest β-APP-IR density at an early point in time post impact. This finding agrees with trends in other studies using the Marmarou model, where large caliber axons of the thoracolumbar region in the spinal cord were found to display their greatest density of positive β-APP at 6 h postinjury, which is earlier than described for brainstem tracts (Czeiter et al., 2008; Marmarou et al., 2005). The CC is composed of a heterogeneous population of unmyelinated (diameter: 0.1–0.7 μm) and myelinated (diameter: 0.3–1.0 μm) axons (Waxman and Black, 1988). This composition may possibly contribute to the wide variation of the CC injury density observed in the present study. Additionally, the CC axons (Waxman and Black, 1988), which are of smaller caliber than Och axons (Duvdevani et al., 1993), exhibited the greatest β-APP-IR density at 28 h post impact. Smaller-caliber axons possess proportionally greater numbers of microtubules per unit area (Friede and Samorajski, 1970; Malbouisson et al., 1985), and larger-caliber axons possess proportionally greater numbers of neurofilaments per unit area (Price et al., 1988). Additionally, an electrophysiology study of compound action potential (CAP) of injured axons in the CC reported the greatest decrease of CAP from fast conducting myelinated fibers
(N1) and slow conducting unmyelinated fibers (N2) at 24 h postTBI (Reeves et al., 2005). Similarly, in our study both β-APP-IR and RMO14-IR axonal densities peaked within the CC at 28 h post-TBI. Also, Reeves et al. (2005) reported that N1 wave amplitude increased at 3 days post-TBI and further increased to a significant recovery by 7 days post-TBI, with no recovery of N2 amplitude. In our study, IR axons in the CC remained present at 7 days post-TBI, suggesting a relation to N2 unmyelinated fiber changes. The quantitative TAI changes in the CC did not show significant differences over time, except between β-APP-IR axonal density at 28 h and 7 days post-TBI; this may be related to the wide variation of the CC injury density among the rats, which has been observed previously (Li et al., 2011a). One study reported that in the ventral brainstem, as time elapsed from 5 min to 60 min, NFC increased and became most prominent at 24 h post-TBI (Povlishock et al., 1997). This was also the case in the present study where RMO14-IR increased from 8 h post-TBI and became most prominent at 28 h post-TBI.
3.3.
Clinical relevance
In this study the peak expression of TAI differed in time for the CC and Och. Also, NFC and IAT presented varying injury
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Fig. 5 – Correlation of RMO14-IR axonal density with β-APP-IR axonal density in the CC (A) and Och (B) for all survival time points. Each circle represents one rat. A strong positive linear correlation was observed between RMO14-IR axonal density and β-APP-IR axonal density in the CC (A) (R2 = 0.831; p < 0.001). Dashed lines represent 95% mean confidence intervals.
density time profiles. These findings suggest that a therapeutic time window is of importance in addressing different pathologies, as it has previously been reported (Loane and Faden, 2010; Xiong et al., 2009). The current findings in CC and Och suggest that the therapeutic time window may vary depending on the WM structures that are affected. Various studies have showed that multiple immunocytochemical approaches are needed to assess the full spectrum of TAI (DiLeonardi et al., 2009; Marmarou et al., 2005; Stone et al., 2001). Initially the accumulation of β-APP occurs in the absence of axolemmal permeability; however, the transport impairment may lead to delayed membrane damage and axolemma permeability (Stone et al., 2004). Treatment targeting IAT may be crucial at earlier time points, as suggested by its peak at 8 h in the Och and 28 h in the CC in this study. Additionally,
horseradish peroxidase investigations have illustrated that axons experiencing axolemma permeability changes will tend to exhibit NFC (Okonkwo et al., 1998; Pettus and Povlishock, 1996; Pettus et al., 1994; Povlishock et al., 1997, 1999). When the axolemmal permeability is altered as a result of TBI, Ca2+ influx and intracellular Ca2+ concentration will increase (Giza and Hovda, 2001; Marmarou et al., 2005; Maxwell and Graham, 1997; Povlishock, 1992; Smith et al., 2003). The increased Ca2+ activates calcineurin (calcium-activated phosphatase) (Hoffman, 1995; Marmarou et al., 2005), which dephosphorylates the neurofilament sidearms, causing NFC. To prevent NFC, various mechanisms can be utilized as treatment. One option is to inhibit the activation of calcineurin by Ca2+, another is to inhibit the dephosphorylation of neurofilament sidearms by calcineurin (Buki et al., 1999b, 2003; Suehiro et al., 2001).
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Our study also suggests that subcellular aspects of TAI occur dependently and/or independently of each other, thereby supporting the use of a multi-drug therapeutic approach to target the full spectrum of TAI.
3.4.
Conclusions
The TAI density was widely variable in the CC and consistent in the Och among the rats. Varying temporal profiles of IAT and NFC were observed in both structures. The density of β-APP-IR axons peaked at 8 h in the CC and 28 h in the Och, whereas the density of RMO14-IR axons peaked at 28 h in both the CC and the Och. After peaking, TAI density decreased over time and remained detectable at 7 days. Additionally, a strong positive linear relationship (R2 = 0.831) between β-APP-IR and RMO14-IR axonal density was observed in the CC but not in the Och. It is suggested that multiple markers are required to fully evaluate TAI. Because each subcellular aspect of TAI demonstrates different profiles over time, utility of diverse pharmacological agents tailored to target various pathological events occurring at different time points may be useful.
4.
Experimental procedure
The institutional animal care and use committee approved all procedures in this study. Twenty-six adult male Sprague Daley rats weighing 394 ± 39.6 g (mean ± SD) (Harlan, IN) were utilized. Twenty-four rats were randomly assigned to four different post-TBI survival time points (n= 6/time point) and two rats were assigned as sham rats. The sham rats were not subjected to surgery and TBI induction. All rats had free access to food and water.
prior to impact and 4 h, 24 h, 3 days and 7 days post impact), each rat was initially anesthetized in a sealed acrylic chamber by a mixture of 2% isoflurane and 0.6 L/min oxygen and maintained under anesthesia for 4 h during the imaging procedure by a mixture of 0.75% to 1.75% isoflurane and 0.6 L/min of oxygen via nose cone. Because the MRI acquisition length was 4 h, the first histological time points in this study were 8 h and 28 h postTBI. At the end of designated survival time (8 h, 28 h, 3 days and 7 days post-TBI), the injured rats were euthanized with an overdose of sodium pentobarbital and exsanguinated. The rats were then transcardially perfused with normal saline followed by cold 4% paraformaldehyde in phosphate buffered saline (0.1 M, pH 7.45). Following perfusion, the brains were carefully removed and post fixed in 4% paraformaldehyde with 20% sucrose. The post fixed brain was cut into a block encompassing the genu and the splenium of the CC which was embedded in Tissue-Tek® O.C.T compound and frozen at −78.5 °C via dry ice. A series of 40 μm coronal frozen sections were then cut from the block (Leica CM 3050, Leica Microsystems GmbH, Heidelberg Germany) and collected in multi-well plates. To represent the Och, two consecutive sections were selected from the anterior, middle and posterior regions of the Och ranging in distance from 0.13 to 0.84 mm posterior to bregma. To represent the CC, three sets of two consecutive sections of 280 μm apart were selected from the caudal portion of the CC. The sections selected to represent TAI in the caudal of the CC were approximately 3–4.5 mm posterior to bregma. Therefore, six sections of the CC and six sections of the Och were processed for β-APP (CC n = 3, Och n = 3), and RMO14 (CC n = 3, Och n = 3) immunocytochemistry analysis.
4.2. 4.1.
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Immunochemistry for IAT and NFC
Surgical preparation and TBI induction
TBI was induced by Marmarou impact acceleration head injury model (Foda and Marmarou, 1994; Marmarou et al., 1994). Initially all rats were anesthetized by 2% to 2.5% isoflurane and 0.6 L/min oxygen in an anesthesia chamber. To induce TBI after initial anesthesia, the rats were maintained with 1% to 1.75% isoflurane and 0.6 L/min oxygen via a nose cone. Then a midline incision was preformed to expose the periosteum covering the vertex of the skull (Marmarou et al., 1994). The periosteum was reflected and a 10 mm diameter steel disk with a thickness of 3 mm affixed by cranioplastic powder (Plastic One, Roanoke, VA) at the midline between the bregma and lamboid sutures. The rats were positioned in a prone position on a foam bed (12×12×43 cm) contained in a Plexiglas box and taped in place around their trunk in order to prevent them from falling off the foam bed after trauma induction. Anesthesia was removed just prior to TBI induction. TBI was induced by dropping a 450 g cylindrical brass weight of 18 mm in diameter from a height of 2 m onto the vertex of the skull at the center of the disk. After the impact, the disk was carefully removed and the skull examined for fractures. Rats with no skull fractures had their skin sutured and were allowed to recover. This study is a part of a parallel imaging study and each rat was maintained under anesthesia for 4 h during the imaging procedure. At the designated imaging time points (at least 1 day
Of the two consecutive sections selected, one section was utilized to stain for β-APP and the other for RMO14. The sections were rinsed 3 × 3 min in phosphate-buffered saline (PBS) and then processed for antigen retrieval by incubation in citrate buffer for 1 h at 75–80 °C. The sections were allowed to cool in the same citrate buffer solution for 30 min and then rinsed 3 × 3 min in PBS. Subsequently, to quench endogenous peroxidase activity the sections were incubated in 0.3% hydrogen peroxidase for 1 h and then rinsed 3 × 3 min in PBS. The brain sections were then incubated overnight either in C-terminus specific APP primary antibody (1 μg/ml; rabbit anti-C-terminus β-APP; cat # 51-2700; Zymed, San Francisco, CA) or in RMO14 primary antibody (1 μg/ml; mouse anti-NF-M; cat # 34-1000; Zymed, San Francisco, CA) with 0.01% Triton X and 1% albumin bovine serum in 2% normal goat serum and PBS. The sections were rinsed 3× 3 min in PBS. The sections that were incubated in APP primary antibody and RMO14 primary antibody were incubated for 2 h in goat anti-rabbit IgG or goat anti-mouse IgG secondary antibody (Vector Laboratories, Burlingame, CA) respectively. The sections were rinsed 3× 3 min in PBS and incubated for 1.5 h in avidin biotin peroxidase complex (Vectastain ABC Standard Elite Kit, Vector). The sections were rinsed 3× 3 min in PBS and briefly incubated in 3, 3′-diaminobenzidine and hydrogen peroxide. Lastly, the brain sections were rinsed 3× 5 min in PBS, dehydrated and cover-slipped using Permount.
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Negative control incubations were performed in the absence of primary antibody.
4.3.
Digital image acquisition
Serial photomicrographs for each coronal section (20× magnification, height= 436.48 μm; length = 327.04 μm; 3.18 pixels/μm) were obtained to encompass the whole region of interest (i.e., Och or CC) by a Zeiss Axio Observer Inverted Microscope (Carl Zeiss, Inc.) fitted with Axio MRC digital camera. The digital images were exported into Adobe Photoshop CS2 for construction of panoramic images of the region of interest (Figs. 6A, D) by photomerging the serial photomicrographs. The saved JPEG panoramic images were imported into ImageJ (http://rsb.info. nih.gov/ij/) to quantify the density of β-APP-IR and RMO14-IR axons.
4.4.
TAI quantification
The panoramic images were viewed in ImageJ on a computer monitor to clearly delineate the Och and CC (Figs. 6B, E). For the Och, the boundaries and the surrounding tissue were digitally removed (Fig. 6C). For the CC, the delineated area was centered on the midline (Fig. 6E) and the tissue outside the delineated area removed (Fig. 6F). The surface area of each delineated region was computed by ImageJ software. These delineated areas of CC and Och were further amplified on the monitor via ImageJ software. Within the delineated areas, the TAI was
quantified by clicking the mouse on the β-APP-IR or RMO14-IR axons, and the cell counter feature in ImageJ software computed the total immunopositive axonal count. β-APP-IR axons that appeared as dense immunopositive swollen profiles and as retraction balls were quantified as injured axons. RMO14-IR axons that appeared as dense immunopositive bands or had a dense punctuated appearance were quantified as injured axons. The injured axonal density was then computed by dividing the sum of the total number of β-APP-IR or RMO14-IR in each panoramic delineated region of interest by the sum of the corresponding delineated areas in square millimeters.
4.5.
Statistical methods
All data were analyzed using SPSS version 17 for Windows. The numbers of injured axons were expressed as the mean value ± standard error of the mean (SEM). The data distribution was tested using the Shapiro–Wilk normality test (p ≤0.05) and Levene's test of homogeneity of variance (p ≤0.05). The mean values of the data set were either not normally distributed as indicated by the Shapiro–Wilk test, or the variances of the data set were heterogeneous as indicated by Levene's test. As the Kruskal–Wallis test does not assume normality in the data set and is insensitive to outliers in the data, this test was utilized to compare the four time points. If the Kruskal–Wallis test showed significance (p≤0.05), then the Mann–Whitney exact test was utilized for pairwise comparisons between two time points. Statistical significance was set at a p value of less than 0.05.
Fig. 6 – Panoramic images of the Och (A) and CC (D). The black outline in Och (B) and CC (E) shows the boundary of the anatomical structures analyzed. Panels C and F represent the area within the outline of the anatomical structure that was analyzed. (20 × magnification) Scale bar of 250 μm.
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Acknowledgments The authors would like to thank Dr. Bulent Ozkan for his advice and guidance on the statistical analysis. The valuable help of Mr. Branden Anderson and Mr. Gurjiwan Virk for data collection is also highly appreciated. Special thanks to Mrs. Charlotte Harman and to Ms. Beth Langelier for editorial assistance.
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