Rapid immunohistological measurement of tyrosine hydroxylase in rat midbrain by near-infrared instrument-based detection

https://doi.org/10.1016/j.jchemneu.2021.101992Get rights and content

Highlights

  • This fresh-frozen tissue IHC approach can be performed rapidly for fast analysis.

  • Tissue is not fixed, allowing simultaneous molecular analysis with fewer animals.

  • Automated quantification of IR secondary allows direct comparison and reproduction.

Abstract

We present a robust, fresh-frozen approach to immunohistochemistry (IHC), without committing the tissue to IHC via fixation and cryopreservation while maintaining long-term storage, using LiCor-based infrared (IR) quantification for sensitive assessment of TH in immunoreacted midbrain sections for quantitative comparison across studies. In fresh-frozen tissue stored up to 1 year prior to IHC reaction, we found our method to be highly sensitive to rotenone treatment in 3-month-old Sprague-Dawley rats, and correlated with a significant decline in rotarod latency-to-fall measurement by approximately 2.5 fold. The measured midbrain region revealed a 31 % lower TH signal when compared to control (p < 0.01 by t test, n = 5). Bivariate analysis of integrated TH counts versus rotarod latency-to-fall indicates a positive slope and modest but significant correlation of R2 = 0.68 (p < 0.05, n = 10). These results indicate this rapid, instrument-based quantification method by IR detection successfully quantifies TH levels in rat brain tissue, while taking only 5 days from euthanasia to data output. This approach also allows for the identification of multiple targets by IHC with the simultaneous performance of downstream molecular analysis within the same animal tissue, allowing for the use of fewer animals per study.

Introduction

Rotenone is a mitochondrial complex I inhibitor which produces systemic inflammation (Liang et al., 2017), alpha-synuclein deposition (Zhang et al., 2017), microglial activation (Sherer et al., 2003a) and loss of motor coordination and tyrosine hydroxylase (TH) positive neurons (Cannon et al., 2009) in the substantia nigra (SN), recapitulating human Parkinson's disease (Betarbet et al., 2000; Monahan et al., 2008; Sherer et al., 2003b) much like other models (Cenci and Lundblad, 2007; Kalinina et al., 2005; Robinson and Becker, 1983; Stenslik et al., 2015; Truong et al., 2006; Zuddas et al., 1994). However, rotenone develops a robust PD phenotype (Anderson et al., 2006; Ferro et al., 2005; Johnson et al., 2018; Schober, 2004). Rotenone has disadvantages in handling and storage because of its toxicity and chemical instability thus it has a short shelf life (Bowman et al., 1978).

Dopamine (DA) production is rate-limited by tyrosine hydroxylase (TH) (Daubner et al., 2011). Dopaminergic neurons of the SN are tonic and their function is dependent upon the post-synaptic receptor (Radulescu, 2010; Sonne and Beato, 2019; Sonne and Lopez-Ojeda, 2019; Swarnkar et al., 2010; Wang et al., 2014). The high biosynthetic activity and mitochondrial dependence of DA production lends itself to rotenone sensitivity (Al-Lahham et al., 2016; Berbusse et al., 2016; Jiang et al., 2019; Lin et al., 2012; Zhang et al., 2018), while dysfunction leads to the motor deficits of PD (Kaasinen et al., 2014; Kelly and Naylor, 1976; Lindner et al., 1999). Furthermore, dopamine loss precedes the loss of TH-positive neurons (Janezic et al., 2013; Sonne, 2013), requiring sub-cellular-level analysis for the detection of early mechanisms of the disorder. The rotarod instrument is commonly used to measure motor coordination in laboratory animals and correlates with TH loss (Faizi et al., 2011; McFadyen et al., 2003; Portmann et al., 2014), but additional and more versatile methods will facilitate the study of early-stage effects in animal models of PD.

Our aim was to study a rodent model of parkinsonism using a sensitive and automated quantification method. The method presented here is robust because of the nature of IR detection, having a wide linear range, and is fast and impartial due to the data collection being performed by an instrument (Shutz-Geschwender et al., 2004).

Section snippets

Materials and methods

The procedure presented here avoids dedicating the entire rat brain tissue to immunohistochemical analysis, allowing for multiple biochemical quantifications to be performed in the same animal. This procedure also reduces the time required to preserve and prepare the rat brain tissue prior to immunohistochemical analysis by avoiding immersion fixation and sucrose cryopreservation prior to sectioning.

Rats were randomly assigned to groups of Untreated, Vehicle-treated, and Rotenone-treated

Results

Motor coordination assessment by rotarod is illustrated in Fig. 1. Latency-to-fall measurements indicate rotenone treatment (RO) results in a 2.5 fold lower rotarod performance when compared with control (CT). (61.0 ± 8.2 vs. 173 ± 15 s, *p < 0.01, n = 5, mean ± SEM). No significant differences were found in untreated vs. vehicle-treated rats, (data not shown) thus they were pooled as CT.

Rotenone treatment suggests a loss of tyrosine hydroxylase (TH) in the midbrain as shown (Fig. 2).

Discussion

Several reports have quantified relative tyrosine hydroxylase levels using near-infrared detection as part of a larger study (Gagnon et al., 2017; Gombash et al., 2013, 2014; Marquez-Valadez et al., 2019; Paumier et al., 2015). However, the lesions were all produced differently from this report. The most common inducer was 6-OHDA injected directly into a region of the nigrostriatal pathway (Gagnon et al., 2017; Gombash et al., 2013, 2014). One study employed rAAV overexpression of α-synuclein (

Conclusions

The methods presented here provide an automated method of quantifying expressed TH levels in sectioned rat brain tissue. We report that the presence of TH signal correlates with motor coordination and show that immunohistochemical detection of TH enzyme by IR-linked secondary antibody may provide a useful measure. We achieve statistical significance with only 5 animals per group, indicating the statistical power of the method using a method without subjective assessment. Our data is presented

Funding

This research received no external funding. This research was funded by University of Central Florida Provost Start-up funds and the Pre-eminent Post-Doctoral Program funding for James W. H. Sonne and Jason S. Groshong. Research was performed at University of Central Florida.

CRediT authorship contribution statement

James W.H. Sonne: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing. Corey Seavey: Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing. Jason S. Groshong: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Supervision, Validation, Visualization,

Declaration of Competing Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Acknowledgments

The authors would like to acknowledge Jason R. Cannon, Ph.D. of Purdue University for many useful discussions regarding the rotenone IP injection rat model of Parkinson's disease and for providing Miglyol-812 N for use in our rotenone solution. We would also like to thank our students Cullen Lemieux and Cheyanne Frosti B.S., M.S., for many useful suggestions and discussions pertaining to this study.

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