Litinetsky L^a; Belinson, H^a; Geva, M^a; Blumenfeld – Katzir, T^b; Loeb, E^c; Amit- Romach, E^a; Orbach, A^a. ^aDiscovery & Product Development, Global Research & Development, Teva Pharmaceutical Industries Ltd., Netanya, Israel; ^b ImagingQ, Ramat Gan, Israel. ^c Patho – Logica Ltd., Golda Meir St 7 ,Scientific Park, Ness Ziona, Israel.

Support for this study was provided by Teva Pharmaceuticals, Israel.

INTRODUCTION
The Cuprizone (CPZ) mouse model is a well-established method for toxic demyelination that has been used in several magnetic resonance imaging (MRI) studies. In this model, mice are fed with CPZ, leading to oligodendrocyte death and subsequent demyelination, most profoundly observed in the corpus callosum (CC). The loss of white matter in the CC can be quantified either by histological tools or imaged by MRI.

MRI analysis is a powerful non-invasive method to evaluate the myelination state of the nervous system and, hence, is highly useful to track the progression of the model and any protective treatment.

The aim of the study was to evaluate the capability of a compact high-performance MRI system (1Tesla aspectimaging®) to assess demyelination of the CC in the cuprizone model.

METHODS
Animals and treatments: Thirty 8-12 week old male C57BL/6 mice were fed for 6 weeks with either normal chow or a diet containing 0.3% cuprizone mixed into pelleted rodent chow ^(1,2). To evaluate if treatment related changes can be detected, we treated mice with test compound X (TCX) which shows beneficial effect in the model and can serve as a positive control. TCX was dosed at 25 mg/kg PO QD. Following 6 weeks of treatment, mice were scarified and perfused in PFA. Brain in suckle were kept prepared for MRI analysis. Thereafter, brains were excised from the skull and prepared for histology. All experimental procedures conformed to accepted ethical standards for use of animals in research and were in accordance with the Committee for the Care and Use of Experimental Animals guidelines and approved by the Teva Institutional Animal Care and Use Committee.

MRI: Ex vivo brain samples were scanned using a 7 Tesla MRI scans (Bruker Biospec 7T/30 Scanner, SCAN@TAU Center) and 1 Tesla (M3^TM compact low MRI machine, aspectimaging®) according to the following protocols:

7 Tesla 1 Tesla

Resolution: 100 micron      Resolution: 100 micron
No. of slices: 12                   No. of slices: 9
Slice thickness: 0.6 mm     Slice thickness: 0.9 mm
Time scan: 26 min            Time scan: 120 min

Assessment of the demyelination of the CC of mice was performed by volumetric analysis of the CC ⁽³⁾

Histopathology: Brains were trimmed in a standard position for all the samples, using the chiasma opticum as a reference landmark. The frontal half brain was processed for paraffin embedding for immunofluorescent staining of MBP and the rest remained in the fixative until cut in a cryostat for Black Gold (BG) staining for myelin.⁽⁴⁾ Stained sections were examined and photographed by using Plan Fluor objectives (40x) connected to a camera (DS-Qi1, Nikon). Digital images were collected and analyzed using Image-Pro software.

* All samples were analyzed by these methods in a blind manner.

RESULTS

1. MRI analysis of loss of CC volume in the CPZ model

Using specific protocols for the 7 Tesla we found that brains from CPZ- treated mice showed a pronounced decrease in CC volume, compared to naive brains (Fig. 1A, B, G). In brains from CPZ-fed mice that were treated with TCX, a drug that ameliorates CPZ-induced demyelination, an intermediate grade of demyelination and CC volume loss was exhibited (Fig. 1C, G). In addition, using unique protocols for the 1 Tesla we were able to measure similar loses in the volume of the CC to those obtained with the 7 Tesla (Fig. 1).

Figure 1: CC volumetric loss is similar in 7 Tesla and 1 Tesla MRI

***p<0.001 – as compared to Naïve group;
### p<0.001 – as compared to CPZ+ Veh, by ANOVA, Fisher’s LSD

1. Histopathology analysis of loss of fibers in the CC in the CPZ model

To further assess the brain pathology we performed MBP immunofluorescent and Black Gold (BG) histological staining. Using BG staining we found that the CC from cuprizone treated mice showed a pronounced decrease in the positive fiber staining, compared to naive brains (Fig. 2A, B, H). In brains from CPZ-fed mice that were treated with TCX, the loss of fibers was significantly ameliorated (Fig. 2C, H). In addition, using MBP immunofluorescent similar results were obtained, by which CPZ treatment decreased MBP positive fibers and TCX treatment ameliorated this effect (Fig. 2G). Important to note, the histological analysis revealed comparable results to those obtained by the MRI analysis of the CC volume loss.

1. Histopathology analysis of loss of fibers in the CC in the CPZ model

To further assess the brain pathology we performed MBP immunofluorescent and Black Gold (BG) histological staining. Using BG staining we found that the CC from cuprizone treated mice showed a pronounced decrease in the positive fiber staining, compared to naive brains (Fig. 2A, B, H). In brains from CPZ-fed mice that were treated with TCX, the loss of fibers was significantly ameliorated (Fig. 2C, H). In addition, using MBP immunofluorescent similar results were obtained, by which CPZ treatment decreased MBP positive fibers and TCX treatment ameliorated this effect (Fig. 2G). Important to note, the histological analysis revealed comparable results to those obtained by the MRI analysis of the CC volume loss.

Figure 2: BG and MBP fiber loss analysis in the CPZ model

***p<0.001 – as compared to Naïve group ;
### p<0.001 – as compared to CPZ+ Veh, by ANOVA, Fisher’s LSD

III. Correlation of MRI and histopathology analysis in the CPZ model

Analysis of the correlation of the MRI and histological analysis revealed that, both MRI 7 Tesla and 1 Tesla correlate similarly with the histological data. Important to note, BG correlation with the MRI analysis seemed to have higher correlation but it was not significantly different.

CONCLUSIONS
The results of the present study show that both MRI methodologies and the histopathology provide comparable readouts for the grade of demyelination of the CC in the cuprizone mouse model. Moreover, the MRI methodologies and the histopathology analysis show significant correlation. Thus, we believe that any of these methodologies could serve as a screen of efficacy in these models. Moreover the same sample could be used for the initial MRI screen and in depth histopathological evaluation.

REFERENCES

1. Torkildsen U, Brunborg LA, Myhr K-M, BL. The cuprizone model for demyelination. ActaNeurol Scand 2008: 117 (Suppl. 188): 72-76.
2. Skripuletz et al. Cortical Demyelination Is Prominent in the Murine Cuprizone Model and Is Strain-Dependent. Am J Pathol 2008, 172 (4): 1053-106.
3. Matsushima, Glenn K., and Pierre Morell. The neurotoxicant, Cuprizone, as a model to study demyelination and remyelination in the central nervous system. Brain Pathology 2001(11.1) : 107-116.
4. Franco-Pons, Neus, et al. Behavioral deficits in the Cuprizone-induced murine model of demyelination/remyelination. Toxicology Letters 2007 (169.3): 205-21.

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Loeb E¹, Zuckerman A¹, Margalit R², Alagem N ³, Sagiv Y ³. ¹Patho-Logica, Golda Meir Street 7, Scientific Park, Ness-Ziona, Israel. ²Science In Action, Sapir Street 5, Scientific park, Ness-Ziona, Israel. ³ Kamada Ltd, Holzman 2, Scientific Park, Rehovot, Israel

Introduction:
The Elastase-induced COPD (chronic obstructive pulmonary disease) mouse model is well-known for emphysema studies. Following administration of test drug, Elastase is instilled intratracheally once a week for four weeks to induce COPD. Lungs are harvested, fixed, and stained for histological evaluation. The H&E-stained slides are analyzed using a digital morphometry tool as applied to the analysis of representative pictures of the affected lung parts.

Aim:
The objective of this study was to develop a reliable quantitative tool for assessing the degree of alveolar loss and rupture damage in treated vs. control lungs of elastase-induced COPD in mice as parameters for the assessment of a treatment effect.

Materials and Methods:

• Anesthetized mice received weekly intratracheal instillations of 20 µL Test Item.
• 24 hours after each dose, 0.2U PPE in 20 µL saline was instilled.
• At the end of weeks 2, 3 & 4, five mice were sacrificed for histology.
• Lungs were fixed in 4% Formaldehyde using intra- and extra-bronchial fixation (gravitation method).
• 5-µm-thick paraffin sections were cut and H&E stained.
• Representative pictures were taken in affected areas (4 fields per lung) using 4X objective magnification

Digital morphometry analysis:
Image analysis was performed using “Image Pro Plus” Version 6.3 (Media Cybernetics, US). From each histological slide, four pictures were taken and sent for digital analysis.

Protocol:

1. 1. Spatial calibration was applied (marked calibration).
2. 2. Area of Interest (AOI) selection was chosen.
3. 3. Threshold selection was based on HSI color system (Hue, Saturation and Intensity).
4. 4. Intensity of 96-255 grey levels was depicted to show the void spaces.
5. The calibrated parameters of Area and Perimeter were measured and used for creating statistical data.

Table 1: Results of morphometry, digital evaluation of lung emphysema.

Figure 1: Illustration of a threshold image measurement of alveolar spaces in the Lung, shown in red X4 H&E.

Results:

• Morphometric quantification shows clear differences in area counts, mean and sum when comparing healthy and affected lungs through accurate comparison between affected lungs (Table 1).
• SD shows greater variation in the measurement of the affected lungs compared to the healthy lungs.
• Areas of air were quantified using a rigid threshold (Fig. 1)
• We extract the numbers from the histological image and show them in a graph for comparative means (Fig. 2)
• Area / Perimeter provide a good index for emphysema quantification (Fig. 3)

Figure 2.  Representative pictures of two normal and two affected lungs with different graph profiles

Figure 3. Summary of x4-fold measurements per animal area / perimeter.

Summary and Conclusions:
We have developed a very accurate and useful tool for the quantification of alveolar loss and rupture damage in a mouse emphysema model. This morphometry technology enables a quantification of the comparison of different treatments effects in this model, and should be applicable to other models where key morphological features can be quantified.

References:

1. Cruz F. F., Antunes M. A., Abreu S. C., Fujisaki L. C., Silva J. D., Xisto D. G., et al. . (2012). Protective effects of bone marrow mononuclear cell therapy on lung and heart in an elastase-induced emphysema model. Respir. Physiol. Neurobiol. 182, 26–36.
2. Weibel E. R. (1990). Morphometry: stereological theory and practical methods, in Models of Lung Disease-Microscopy and Structural Methods, eds Gil J., editor. (New York, NY: Marcel Dekker; ), 199–247

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Loeb E¹, Zuckerman A¹, Margalit R², Orgad U³. ¹Patho-Logica, Golda Meir Str 7, Scientific Park, Ness-Ziona, Israel. ²Science In Action, Sapir Str. 5, Scientific park, Ness-Ziona, Israel. ³Hebrew University, Koret School for veterinary medicine, Rehovot, Israel

Introduction:

• Parkinson’s disease is a progressive neurodegenerative disorder, that is primarily characterized by degeneration of the dopaminergic neurons of the nigrostriatal pathway.
• The neurotoxin 6-hydroxydopamine (6-OHDA) continues to constitute a valuable tool to model Parkinson’s disease in rats and mice (1).

Aim:

• To quantify the amount of damaged dopamine producing cells and fibers by digital morphometric tools, compared to scale traditional semi-quantitative analysis based on score scale (2).

Materials and methods:

• Intrastriatal  injection of  6-OHDA (3 μl) was applied into the right hemisphere of mice (n=5), at the following coordinates: (i) AP=+1.0, L=−2.1, DV=−2.9;(3). The left hemisphere served as control.
• Animals were sacrificed 8 weeks later. Brains were dissected to obtain sections from the striatum (ST) and the Substantia Nigra Pars Compacta (SNC).
• Five mµ thick paraffin sections were cut and stained for tyrosine hydroxylase (TH) activity, a marker for dopamine production, using immunofluorescence histochemistry.
• The intensity of the reaction was measured by computerized morphometry (Image Pro Plus 6.3), and compared to the semi-quantitative analysis, (score scale).

Materials and methods:

• Intrastriatal  injection of  6-OHDA (3 μl) was applied into the right hemisphere of mice (n=5), at the following coordinates: (i) AP=+1.0, L=−2.1, DV=−2.9;(3). The left hemisphere served as control.
• Animals were sacrificed 8 weeks later. Brains were dissected to obtain sections from the striatum (ST) and the Substantia Nigra Pars Compacta (SNC).
• Five mµ thick paraffin sections were cut and stained for tyrosine hydroxylase (TH) activity, a marker for dopamine production, using immunofluorescence histochemistry.
• The intensity of the reaction was measured by computerized morphometry (Image Pro Plus 6.3), and compared to the semi-quantitative analysis, (score scale).

Semi-quantitative analysis (score scale):

–   = Normal TH expression
+   = Mild TH reduction
++ = Moderate reduction
+++ = Severe TH reduction

Table 1: Semi quantitative analysis of TH activity based on a score scale. Note the reduction of TH in the treated side (R) compared with the untreated hemisphere (L).

Table 2: Quantitative analysis of TH activity using digital morphometry. Note the decrease in the treated side (R) compared with the untreated hemisphere (L).

Table 3: Histogram of % of TH positive cells in SNC

Results:

• Morphometric analysis: The quantitative analysis showed a pronounced decrease in the intensity of the reaction in the ST in the right hemisphere, compared to the left un-treated control.
• In the SNC, the activity of TH in the right hemisphere was lower compared to the control left hemisphere.
• Semi quantitative analysis: A decrease  of TH in the ST and SNC was observed, compared to the left hemisphere.
• The morphometric analysis could detect slight changes that were not shown by the semi-quantitative analysis (for example see animal 181).

Figure 1. Coronal brain sections at the level of the striatum, showing the activity of TH expressed by fluorescence. Note the reduction in TH expression in the right hemisphere compared to the left control. The digital Intensity analysis is presented in the histogram below.

Figure 2. Coronal brain sections at the level of the substantia nigra, showing the intensity of TH activity. Note the reduction of TH expression (fluorescence) in the right hemisphere, compared to the left side.  The morphometric digital surface analysis of TH positive cells is presented in the left image.

Summary and Conclusions:

• A clear damage in the striatum and in the substatia nigra was shown following injection of 6-OHDA , expressed by decrease in TH activity.
• An accurate number for the TH activity was obtained, by using a digital morphometric tool, in contrast to the semi-quantitative method.
• The digital quantification of histological markers is highly accurate compared to the traditional semi-quantitative method and therefore recommended especially for efficacy studies, where slight differences between treatments are essential.

References:

1. Schober A. Classic toxin-induced animal models of Parkinson’s disease: 6-OHDA and MPTP, Cell & Tissue Res 2004; 318: 215–224.
2. Jansen JA et al. Semi-quantitative and qualitative histologic analysis method for the evaluation of implant biocompatibility.  J Invest Surg; 1994,;7:123-34.
3. Fischer K et al. In vivo quantification of dopamine transporters in mice with unilateral 6-OHDA lesions using [11C]methylphenidate and PET. Neuroimage 2012; 59:2413-22.
4. Mori S et al. Lack of interleukin-13 receptor α1 delays the loss of dopaminergic neurons during chronic stress. J Neuroinflammation 2017: 14:88.

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