PhD in Veterinary Sciences for Animal Health and Food Safety

Contacts

Supervisor

Antonio D'angelo

Phd thesis

Project title
Biomarkers' identification in cerebrospinal fluid: new approaches for discriminating Central Nervous System disorders in veterinary neurology

Scientific background
Central nervous system (CNS) disorders represent a broad spectrum of diseases of different ethiologies. The therapeutic approach is extremely different for each of these conditions, and the indicated therapy for a specific CNS disease may be critically detrimental for a different one. Cerebrospinal fluid (CSF) surrounds and permeates the CNS. In patients with CNS disorders, the CSF is routinely analyzed for its cellular and chemical composition, and for specific antibodies and infectious agents research, representing a mainstay for the diagnosis of inflammatory CNS disorders. However, current techniques for the CSF analysis do not have high specificity in discriminate different CNS disorders, and for this reason developing other CSF biomarkers is fundamental to further improve the diagnostic value of this analysis.

When the nervous tissue is injured in the course of a CNS pathology, immune system cells, especially different sub-types of CD4 T-cells, produce and release inflammatory mediators such as cytokines, chemokines and growth factors in the CSF, with particular patterns based on the sub-type of cells activated by each etiopathogenetic mechanism.
These different CSF patterns have been evaluated in human medicine by the Luminex multiplex assay (Luminex) technology, suggesting their utility as biomarkers in early diagnosis, prognosis, and monitoring process. Currently, in veterinary literature cytokines and chemokines CSF profile has been evaluated only in association with certain CNS disorders with promising results.

Another extremely promising biomarker studied in human medicine is the Neurofilament light chain (NfL), one of three subunits of the Neurofilament (NFs) that compose the axonal cytoskeleton. NFs levels increase within the CSF and the blood after axonal damage, so they may be used as biomarker able to quantify neuronal damage in various CNS diseases even before the occurrence of severe clinical signs, to predict probable disorder progression and to monitor the effects of therapies. The current reference method of NfL quantification is
the Single-molecule array (Simoa) technology, but recently the Ella automated immunoassay system (Ella) has been proposed as a valid alternative, allowing the evaluation of a higher number of samples, in less time and with overall lower costs.
In veterinary medicine, normal plasma NfL concentration and its correlation with age, stature and body weight have been established in dogs. Moreover, the diagnostic and monitoring role of NfL has been evaluated in course of canine meningoencephalitis of unknown origin and certain neurodegenerative disorders, while no studies have been conducted in cattle.

Aims
Based on these premises, the aim of the present PhD project is to evaluate specific CSF patterns of cytokines and chemokines and CSF and blood NfL concentration in in course of bovine and canine CNS disorders. The main and primary objective is to improve the possibilities of a prompt diagnosis, especially the capability to discriminate among different CNS diseases, with mild invasive procedures such as blood and CSF collection and analysis. An early identification of the ongoing pathology is essential to promote an appropriate and rational usage of drugs, and to reduce the mortality associated with these conditions.

Specifically, the research project will consist of the sequent objectives:

NfL
- Define serum and CSF NfL concentrations in healthy cattle with Ella (Auth. n° 242/2020 – PR);
- Quantify serum and CSF NfL levels in neurological bovine and canine patients referred to the Neurology Service of the Veterinary Teaching Hospital (VTH) of Turin with Ella;
- Carry out Simoa tests on both healthy and neurological patients, comparing the results with Ella to assess the agreement between the two techniques;
- Assess the feasibility of Ella analysis for -20°C stored CSF and serum samples, by comparing the results with those from -80°C stored samples;               
- Investigate the correlation between the results obtained from serum and CSF samples.

Cytokines/chemokines
- Evaluate a selected panel of cytokines and chemokines on CSF samples collected from bovine and canine patients referred to the Neurology Service of the Veterinary Teaching Hospital (VTH) of Turin with a CNS disease with Luminex and determine their diagnostic utility in differentiating CNS disorders;

Preliminary results

NfL
Ella analyses were conducted on 33 CSF and 19 serum samples from healthy cattle (Auth. n° 242/2020 – PR) and on 96 CSF and 29 serum samples from cattle with CNS disorders, classified under the VITAMIN D acronym (Vascular, Infectious/inflammatory, Traumatic, Anomaly, Metabolic/toxic, Idiopathic, Neoplastic, Degenerative).
Due to the correlation between NfL and age, bovines included were categorized as calves (≤ 2 months) and juvenile cattle (2-12 months). CSF NfL levels were established for each healthy group (calves: median=837 pg/ml; interquartile range=504-1157 pg/mL - juvenile cattle: median=361,5 pg/mL; interquartile range=280,75-528,75 pg/mL), while serum NfL concentration was assessed only for juvenile cattle (median: 6.31 pg/mL; interquartile range: 5.26-8.97 pg/mL). CSF NfL levels were quantified also for each pathological group and compared to healthy animals of the same age, revealing significantly higher CSF NfL levels in calves with degenerative disorders (p=0.017), and in juvenile cattle with infectious/inflammatory conditions (p<0.001) and with metabolic/toxic diseases (p=0.002).
Duplicate analysis of samples stored at both -20°C and -80°C showed no significant temperature-related differences in NfL concentration (CSF: rho=0.91, p<0.001; serum: rho=0.94, p<0.001).
A significant positive correlation (rho=0.5, p<0.001) between serum and CSF NfL levels was found, suggesting the potential for less invasive blood sampling in patient monitoring.

Cytokines/chemokines
A preliminary Luminex test was performed on 36 bovine CSF samples (8 healthy, 28 with CNS disorders) using the MILLIPLEX Bovine Cytokine/Chemokine Magnetic Bead Panel 1, preformed with 15 analytes (IFNγ, IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17A, IL-36RA, IP-10, MCP-1, MIP-1α, MIP-1β, TNFα, VEGF-A). 9 analytes (IFNγ, IL-1α, IL-6, IL-8, IL-10, IL-36RA, IP-10, MCP-1, VEGF-A) were selected for future tests, excluding low-expressing cytokines/chemokines.

References
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Dewey CW, Dacosta RC. Practical Guide to Canine and Feline Neurology. Wiley Blackwell; 2016. 687 p.

Yuan A, Nixon RA. Neurofilament Proteins as Biomarkers to Monitor Neurological Diseases and the Efficacy of Therapies. Front Neurosci. 2021 Sep 27;15:689938.

Zelek WM, Fathalla D, Morgan A, Touchard S, Loveless S, Tallantyre E, et al. Cerebrospinal fluid complement system biomarkers in demyelinating disease. Mult Scler. 2020 Dec;26(14):1929–37.

d’Abramo C, D’Adamio L, Giliberto L. Significance of Blood and Cerebrospinal Fluid Biomarkers for Alzheimer’s Disease: Sensitivity, Specificity and Potential for Clinical Use. JPM. 2020 Sep 8;10(3):116.

Olsson B, Portelius E, Cullen NC, Sandelius Å, Zetterberg H, Andreasson U, et al. Association of Cerebrospinal Fluid Neurofilament Light Protein Levels With Cognition in Patients With Dementia, Motor Neuron Disease, and Movement Disorders. JAMA Neurol. 2019 Mar 1;76(3):318.

Peng L, Bi C, Xia D, Mao L, Qian H. Increased cerebrospinal fluid neurofilament light chain in central nervous system inflammatory demyelinating disease. Multiple Sclerosis and Related Disorders. 2019 May;30:123–8.

Disanto G, Barro C, Benkert P, Naegelin Y, Schädelin S, Giardiello A, et al. Serum Neurofilament light: A biomarker of neuronal damage in multiple sclerosis: Serum NfL as a Biomarker in MS. Ann Neurol. 2017 Jun;81(6):857–70.

Kothur K, Wienholt L, Brilot F, Dale RC. CSF cytokines/chemokines as biomarkers in neuroinflammatory CNS disorders: A systematic review. Cytokine. 2016 Jan;77:227–37.

Kuhle J, Disanto G, Lorscheider J, Stites T, Chen Y, Dahlke F, et al. Fingolimod and CSF neurofilament light chain levels in relapsing-remitting multiple sclerosis. Neurology. 2015 Apr 21;84(16):1639–43.

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Dung Le N, Muri L, Grandgirard D, Kuhle J, Leppert D, Leib SL. Evaluation of neurofilament light chain in the cerebrospinal fluid and blood as a biomarker for neuronal damage in experimental pneumococcal meningitis. J Neuroinflammation. 2020 Dec;17(1):293.

Nötzel M, Werder LI, Ziemssen T, Akgün K. Ella versus Simoa Serum Neurofilament Assessment to Monitor Treatment Response in Highly Active Multiple Sclerosis Patients. IJMS. 2022 Oct 15;23(20):12361.

Gauthier A, Viel S, Perret M, Brocard G, Casey R, Lombard C, et al. Comparison of SimoaTM and EllaTM to assess serum neurofilament‐light chain in multiple sclerosis. Annals of Clinical and Translational Neurology. 2021 Apr 8;8(5):1141–50.

Perino J, Patterson M, Momen M, Borisova M, Heslegrave A, Zetterberg H, et al. Neurofilament light plasma concentration positively associates with age and negatively associates with weight and height in the dog. Neuroscience Letters. 2021 Jan;744:135593.

Yun T, Koo Y, Chae Y, Lee D, Kim H, Yang M, et al. Association between neurofilament light chain concentration and lesion size in dogs with meningoencephalitis of unknown origin. Veterinary Medicine & Sci. 2023 Jul;9(4):1541–6.

Yun T, Koo Y, Chae Y, Lee D, Kim H, Kim S, et al. Neurofilament light chain as a biomarker of meningoencephalitis of unknown etiology in dogs. J Vet Intern Med. 2021 Jun 10;Online ahead of print.

Panek WK, Gruen ME, Murdoch DM, Marek RD, Stachel AF, Mowat FM, et al. Plasma Neurofilament Light Chain as a Translational Biomarker of Aging and Neurodegeneration in Dogs. Mol Neurobiol. 2020 Jul;57(7):3143–9.

Fortuna D, Hooper DC, Roberts AL, Harshyne LA, Nagurney M, Curtis MT. Potential role of CSF cytokine profiles in discriminating infectious from non-infectious CNS disorders. Horwitz MS, editor. PLoS ONE. 2018 Oct 31;13(10):1–17.

Barber RM, Platt SR, De Risio L, Barber J, Robinson KR. Multiplex analysis of cytokines in the cerebrospinal fluid of dogs after ischemic stroke reveals elevations in chemokines CXCL1 and MCP-1. Front Vet Sci. 2023 May 17;10:1169617.

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Willems N, Tellegen AR, Bergknut N, Creemers LB, Wolfswinkel J, Freudigmann C, et al. Inflammatory profiles in canine intervertebral disc degeneration. BMC Vet Res. 2016 Dec;12(1):10.

Martin-Vaquero P, da Costa RC, Moore SA, Gross AC, Eubank TD. Cytokine Concentrations in the Cerebrospinal Fluid of Great Danes with Cervical Spondylomyelopathy. J Vet Intern Med. 2014 Jul;28(4):1268–74.

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Burgener I, Van Ham L, Jaggy A, Vandevelde M, Tipold A. Chemotactic activity and IL-8 levels in the cerebrospinal fluid in canine steroid responsive meningitis–arteriitis. Journal of Neuroimmunology. 1998 Aug;89(1–2):182–90.

 

Research activities

Attended courses and congress

35^th ESVN-ECVN Symposium, 21^st-23^rd September 2023, Venezia, Italy

SINVET Meeting “Le malattie infettive del Sistema Nervoso”, 15^th April 2023, online meeting

Doctoral School course "Open Science A to Z", 17^th-18^th April 2023, Torino, Italy

Doctoral School course "Bibliography and bibliometrics like pros: Stay up to date and Develop Literature Syntheses in Biomedical Domain", 1^st February-18^th May 2023, online course

Pratical course by University of Parma "IV Corso avanzato di Neuroelettrodiagnostica del cane e del gatto", 19^th-20^th November 2022, Parma, Italy

SINVET Meeting “Le ernie del disco: un mondo in continua evoluzione”, 25^th-26^th November 2022, Cremona, Italy

34^th ESVN-ECVN Symposium, 22^th-24^th September 2022, Palma de Mallorca, Spain

Grant Writing Course - How to write a Competitive Application to a Funding Agency, 5^th-9^th September 2022, Turin, Italy

Practical course by SCIVAC "Neurodiagnostica RM", 30^th June-2^nd July 2022, Cremona, Italy

Live webinar by ESAOTE "MRI of CNS Vascular diseases by Dr. Cristian Falzone", 27^th April 2022, online meeting

SINVET Meeting with Dennis O'Brien, 9^th April 2022, online meeting

Theoretical-practical course "Filmmaking for Scientists", 13^th-17^th December 2022, Turin, Italy 

SINVET Meeting “Diagnostica per immagini in neurologia veterinaria: quanto avanzata?”, 27^th November 2021, online meeting

Publications
All of my research products