Regional calcineurin subunit B isoform expression in rat hippocampus following a traumatic brain injury

Abstract

Calcineurin subunit isoforms are implicated in long term potentiation, long term depression, and structural plasticity. Calcineurin inhibitors benefit axonal damage, cellular dysfunction, and cognitive outcomes in animal models of traumatic brain injury (TBI). Distribution of the catalytic calcineurin A subunit is altered and calcineurin activity increased following fluid percussion injury. Alterations in calcineurin subunit A isoform distribution within the hippocampus also occur post controlled cortical impact (CCI) demonstrating a reduction in catalytic subunit distribution in CA1-2 dendritic fields. Furthermore the effect of TBI on the regulatory subunit, calcineurin B, is unknown. Understanding the role of both subunits is necessary to effectively target alterations in calcineurin signaling as current calcineurin inhibitors, such as cyclosporin A and FK-506, rely upon binding sites on both subunits for complete inhibition. The effect of moderate CCI on the expression and distribution of calcineurin B isoforms within the hippocampus was examined at 2h and 2weeks post injury. Calcineurin B isoforms showed increased expression throughout the CA1 and CA2 while there was a decrease in expression within the ipsilateral dentate gyrus. Alterations in CnB isoform expression within the CA1, CA1-2, and dentate gyrus have significant implications for persistent hippocampal dysfunction following TBI. Regional changes in regulatory subunit expression may alter the effect of calcineurin inhibitors regionally following a traumatic brain injury.

Figure 1

CnB1 immunohistochemistry demonstrating alterations in CnB1 isoform distribution 2 hours following TBI (right panels). Arrows indicate side of cortex ipsilateral to injury or sham surgery. There is a noticeable increase of staining within CA1 and CA1-2 in TBI (1a and 2a) versus sham (1 and 2). There is a significant loss of CnB1 expression within the EB of the DG in TBI (4a) versus sham (4). Scale bars, 50 µm for regional sections. Abbreviations: SO= Stratum Oriens; SP= Stratum Pyramidale; SR= Stratum Radiatum; SL= Stratum Lucidum; SM= Stratum Moleculare; SG= Stratum Granulosum; HB= Hidden Blade; H=Hilus; EB= Exposed Blade.

Figure 2

Representative higher power images demonstrating the increase of CnB1 isoform staining in CA1 of TBI animals (B – white arrow) compared to sham (A – white arrow) at 2 hours post injury, with a relative sparing of dendrites (A and B – black arrows). There is also a noticeable decrease in the exposed blade of the dentate in the SG cell body layer as seen in TBI animals (D – white arrow) compared to sham (C – white arrow). There appears to be some cell loss in the dentate which may explain some of the decreases with both isoforms. Similar results can be seen at 2 weeks for CnB1 and at 2 hours (Figure 6) and 2 weeks post injury for CnB2.

Figure 3

CnB1 immunohistochemistry demonstrating alterations in CnB1 isoform distribution 2 weeks following TBI (right panels). Arrows indicate side of cortex ipsilateral to injury or sham surgery. There is an increase in staining within the CA1, CA1-2, and CA3 in TBI (1a, 2a, 3a) versus sham (1, 2, 3). There is a significant loss of CnB1 expression within the EB of the DG in TBI (4a) versus sham (4). Scale bars, 50 µm for regional sections. Abbreviations: SO= Stratum Oriens; SP= Stratum Pyramidale; SR= Stratum Radiatum; SL= Stratum Lucidum; SM= Stratum Moleculare; SG= Stratum Granulosum; HB= Hidden Blade; H=Hilus; EB= Exposed Blade.

Figure 4

CnB2 immunohistochemistry demonstrating alterations in CnB2 isoform distribution 2 hours following TBI (right panels). Arrows indicate side of cortex ipsilateral to injury or sham surgery. There is a significant increase in staining within the CA1 and CA1-2 in TBI (1a and 2a) versus sham (1 and 2). There is a significant loss of CnB2 expression within the EB of the DG in TBI (4a) versus sham (4). Scale bars, 50 µm for regional sections. Abbreviations: SO= Stratum Oriens; SP= Stratum Pyramidale; SR= Stratum Radiatum; SL= Stratum Lucidum; SM= Stratum Moleculare; SG= Stratum Granulosum; HB= Hidden Blade; H=Hilus; EB= Exposed Blade.

Figure 5

Representative higher power images demonstrating the increase of CnB2 isoform staining in the deep SR of TBI animals (B – white arrow) compared to sham (A – white arrow) at 2 hours post injury, with a relative sparing of dendrites (A and B – black arrows). Unlike CnB1 (Figure 2), the decrease in the exposed blade of the dentate in the SG cell body layer in TBI animals (D – white arrow) compared to sham (C – white arrow) is not as striking.

Figure 6

CnB2 immunohistochemistry demonstrating alterations in CnB2 isoform distribution 2 weeks following TBI (right panels). Arrows indicate side of cortex ipsilateral to injury or sham surgery. There is a significant increase in CnB2 isoform expression within both the CA1 and CA1-2 regions in injured (1a and 2a) compared to shame (1 and 2). There is a significant loss of CnB2 expression within the EB of the DG in TBI (4a) versus sham (4). Scale bars, 50 µm for regional sections. Abbreviations: SO= Stratum Oriens; SP= Stratum Pyramidale; SR= Stratum Radiatum; SL= Stratum Lucidum; SM= Stratum Moleculare; SG= Stratum Granulosum; HB= Hidden Blade; H=Hilus; EB= Exposed Blade.

Figure 7

Western blot analysis of specific hippocampal regions. Homogenates were made from pooled resection tissue from N=3 rats per group. There is a noticeable increase in both CnB1 and CnB2 expression within the CA1 region at both timepoints examined. There is also a noticeable decrease in both CnB subunit isoforms expression within the DG of injured animals compared to sham, which is consistent with immunohistochemistry. Results of western blot analysis of the CA2-CA3 region do not completely agree with immunohistochemistry suggesting that there may be an alteration in distribution within these regions as well as changes in total expression. Abbreviations: Sh-C= Sham Contralateral; TBI-C= Traumatic Brain Injury Contralateral; Sh-I= Sham Ipsilateral; TBI-I= Traumatic Brain Injury Ipsilateral.