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 (Nf-L), 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 Nf-L 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 Nf-L concentration and its correlation with age, stature and body weight have been established in dogs. Moreover, the diagnostic and monitoring role of Nf-L has been evaluated on serum/plasma in course of canine structural brain disorders, epilepsy 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 Nf-L 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:

Nf-L levels in healthy and diseased cattle
- Assess the stability of Nf-L at different storage temperature (-20°C/-80°C);
- Define serum and CSF Nf-L concentrations in healthy cattle with Ella (Auth. n° 242/2020 – PR);
- Quantify CSF Nf-L levels in neurological bovine patients referred to the Neurology Service of the Veterinary Teaching Hospital (VTH) of Turin with Ella;             
- Investigate the relationship between the serum and CSF Nf-L levels.

Nf-L levels in diseased dogs
- Assess the stability of Nf-L at different storage temperature (-20°C/-80°C);
- Quantify CSF Nf-L levels in neurological canine patients referred to the Neurology Service of the Veterinary Teaching Hospital (VTH) of Turin with Ella;             
- Investigate the relationship between the serum and CSF Nf-L levels.

Cytokines/chemokines in healthy and diseased cattle
- Characterize CSF cytokines/chemokines pattern of expression in healthy cattle and cattle affected by CNS disorders referred to the Neurology Service of the Veterinary Teaching Hospital (VTH) of Turin with Luminex;
- Investigate the role of CSF cytokines/chemokines in distinguishing between bovine CNS disorders.

Results and discussion
Nf-L levels in healthy and diseased cattle
Ella analyses were conducted on 49 CSF and 39 serum samples from healthy cattle (Auth. n° 242/2020 – PR) and on 75 CSF and 32 serum samples from cattle with CNS disorders, classified under the VITAMIN D acronym (Vascular, Infectious/inflammatory, Traumatic, Anomaly, Metabolic/toxic, Idiopathic, Neoplastic, Degenerative).

Assess the stability of Nf-L at different storage temperature (-20°C/-80°C)
The initial aim was to assess Nf-L stability at different storage temperatures, as 29 CSF samples and 15 serum samples were retrospectively enrolled in the project an they were only stored at -20°C. Despite human studies suggesting Nf-L stability across multiple freeze-thaw cycles and extended room temperature exposure, current guidelines recommend -80°C storage for CSF. To verify the viability of these samples, a comparison of Nf-L levels in paired samples stored at -20°C and -80°C was performed using Spearman’s rank correlation and the Wilcoxon signed-rank test. The strong correlation and lack of significant differences between storage conditions allowed the inclusion of samples stored at -20°C in subsequent analyses, thus broadening the dataset.

Define serum and CSF Nf-L concentrations in healthy cattle (Auth. n° 242/2020 – PR)
CSF and serum Nf-L levels in healthy animals were defined for the first time. Drawing on human research, where Nf-L levels correlate with age due to neuronal maturation and aging processes, potential associations between age and Nf-L levels in cattle were analyzed. Healthy cattle were divided into two subgroups: calves under 1 year and adult cattle over 1 year. Statistical analysis, including simple linear regression, revealed no age-related association in calves’ serum, a weak association in calves' CSF and a moderate to strong association in adult cattle's serum and CSF. These findings align partly with human data, but the small sample size, particularly in calves, limited the conclusiveness of this observation.

Quantify CSF Nf-L levels in neurological bovine patients 
The third and key aim of this section was to evaluate CSF Nf-L levels in neurologically impaired cattle, assessing its potential as a diagnostic biomarker. To do this, Nf-L levels were compared between healthy and diseased cattle (categorized according to the VITAMIN D acronym), highlightening significantly higher CSF Nf-L levels in cattle with degenerative and infectious disorders compared to healthy controls.

Degenerative disorders are uncommon conditions in cattle, therefore all the animals included in this group were affected by the same disorder, the spinal muscular atrophy (SMA). This makes it difficult to generalize these findings beyond the specific disorder studied.

On the other hand, infectious CNS conditions are common in cattle, with different pathogens affecting various age groups. In human medicine, there are controversial evidence regarding the use of Nf-L concentration to discriminate between etiological agents of CNS infections, however a recent study reported higher CSF Nf-L levels in patients with pneumococcal meningitis than in those with non-pneumococcal meningitis. Despite limitations in pathogen identification, a subset of calves with confirmed E. coli meningoencephalomyelitis showed significantly higher Nf-L levels compared to healthy cattle. These findings suggest a role for Nf-L in diagnosing specific infections, but more research is needed to distinguish between different pathogens.

For metabolic/toxic and congenital CNS anomalies, no significant differences in Nf-L levels were observed compared to healthy cattle. This outcome was expected, particularly in congenital anomalies where ongoing axonal damage is not anticipated.

CSF Nf-L levels were also compared between different groups of diseased animals, with similar results. CSF Nf-L levels resulted significantly higher in calves affected by infectious and degenerative conditions when compared with CNS anomalies and in juvenile cattle affected by infectious disorders when compared with those affected by metabolic/toxic ones. To further understand Nf-L's diagnostic potential, the area under the ROC curve was calculated to establish optimal cutoff values for distinguishing between CNS diseases.
          
Investigate the relationship between the serum and CSF Nf-L levels
The use of serum Nf-L as a proxy for CSF levels, a non-invasive approach commonly used in human medicine, was also explored. Simple linear regression analysis revealed only a weak relationship between serum and CSF Nf-L levels in diseased cattle, suggesting that serum may not be a reliable substitute for CSF in this context.

Nf-L levels in diseased dogs
Ella analyses were conducted on 83 CSF and 17 serum samples from dogs affected by CNS disorders, classified under the VITAMIN D acronym (Vascular, Infectious/inflammatory, Traumatic, Anomaly, Metabolic/toxic, Idiopathic, Neoplastic, Degenerative).

Assess the stability of Nf-L at different storage temperature (-20°C/-80°C)
Due to the retrospective nature of some samples stored at -20°C, their viability was assessed. While CSF Nf-L levels remained stable across storage conditions, a significant difference was noted in serum samples, likely due to pre-analytical factors such as accidental thawing. As a result, these serum samples were excluded from further analysis.

Quantify CSF Nf-L levels in neurological canine patients
As first aim, the Nf-L role in the course of intracranial idiopathic disorders was assessed. This are disorders with no identifiable cause that usually requires a diagnosis of exclusion. Idiopathic disorders are very common in dogs, and in veterinary literature Nf-L levels in the course of these conditions are comparable to those of healthy dogs, as a primary damage to neurons is not present. However, this evidence was only explored on serum in dogs. CSF Nf-L levels from idiopathic dogs were compared with data about CSF Nf-L levels in healthy dogs taken from the literature. A significant difference between them was not found, confirming what observed on serum.

In examining CSF Nf-L levels in dogs with intracranial disorders, it was found that Nf-L levels were significantly higher in dogs with structural disorders compared to those with idiopathic conditions, and the optimal cut-off value to distinguishing between them was calculated. However, Nf-L levels were not able to discriminate among the different VITAMIN D categories belonging to structural disorders. Among epileptic dogs, those with structural epilepsy had higher Nf-L levels than those with idiopathic epilepsy, and the optimal cut-off value to distinguishing between them was calculated. A possible impact of seizure activity on Nf-L levels was investigate, but an association between Nf-L levels and the time from the last seizure was not observed.

For spinal cord disorders, significant differences in CSF Nf-L levels across VITAMIN D categories could not be established. 
            
Investigate the relationship between the serum and CSF Nf-L levels
Analysis regarding the potential use of serum Nf-L as a proxy for CSF Nf-L in dogs revealed only a weak relationship between serum and CSF Nf-L levels, suggesting that at the moment serum measurements should be interpreted cautiously.

Cytokines/chemokines in healthy and diseased cattle
A preliminary Luminex test was performed on 36 bovine CSF samples (8 healthy, 28 with CNS disorders) using the Multiplex 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 cytokines/chemokines fairly/not expressed by samples.

Characterize CSF cytokines/chemokines pattern of expression in healthy cattle and cattle affected by CNS disorders 
Luminex analyses were conducted on 86 CSF samples, 30 from healthy cattle (Auth. n° 242/2020 – PR) and 56 from cattle affected by CNS disorders, classified under the VITAMIN D acronym (Vascular, Infectious/inflammatory, Traumatic, Anomaly, Metabolic/toxic, Idiopathic, Neoplastic, Degenerative).

For the first time, the cytokine and chemokine expression profile in healthy cattle was characterized, revealing a pattern similar to that previously reported in healthy dogs.

Cytokines/chemokines levels measured in diseased cattle were normalized using healthy samples and a clustering was implemented for both cytokines and cattle's etiological categories. With these data, it was created an heatmap that showed a clear division of cattle into four subgroups. A first subgroup was a large heterogeneous subgroup in which the majority of evaluated cytokines showed no differences in expression compared to healthy cattle. The other three subgroups consisted mainly of infectious/inflammatory cases, with some cytokines showing increased expression than in healthy controls while other were less expressed.

Investigate the role of CSF cytokines/chemokines in distinguishing between bovine CNS disorders
To verify the information obtained from the heatmap and assess the presence of statistically significant differences in cytokines pattern of expression in diseased cattle, CSF levels of each cytokine measured in healthy cattle and cattle affected by CNS disorder categorized according to the VITAMIN D acronym were compared. The analysis revealed no significant differences in the cytokine profile of cattle affected by traumatic, vascular, metabolic/toxic, or congenital disorders. However, significant differences were observed in cattle with infectious/inflammatory conditions. Specifically, IL-6 and MCP-1 levels were significantly elevated, while IL-10 and IL-36RA levels were reduced compared to healthy controls. These findings align with known biological mechanisms, where IL-6 and MCP-1 are involved in inflammation and immune cell recruitment, whereas IL-10 and IL-36RA act to inhibit inflammation.

These findings suggest a promising role for cytokine quantification in the CSF as a diagnostic tool for neurological diseases in cattle. However, the limited sample size in certain VITAMIN D categories, such as metabolic/toxic, traumatic, vascular, and congenital disorders, indicates that further studies with larger cohorts are needed.

Conclusion
This study contributed to the understanding of cytokine/chemokines and Nf-L dynamics in cattle and dogs. It revealed the diagnostic potential of CSF cytokine/chemokines and Nf-L analysis, although they don't represent a substitute for standard CSF analysis. However, it's uncommon that a single biomarker provides all the infornation needed, but these instrument together can contribute to reach the goal of a more accurate diagnosis.

References
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Research activities

Oral presentations and posters
Neurofilament light chain (Nf-L) quantification in cerebrospinal fluid and serum as
a possible biomarker in the differential diagnosis of bovine neurological diseases
Di Muro, G.; Tessarolo, C.; Cagnotti, G.; Favole, A.; Ferrini, S.; Ala, U.; Bellino, C.; Borriello,
G.; Palmitessa, C.; Iamone, G.; Iulini, B.; Pezzolato, M; Corona, C.; D'angelo, A.
Poster and Oral Presentation at 36^th ESVN-ECVN Symposium
13^th – 14^th September 2024, Porto, Portugal

Quantificazione della catena leggera del neurofilamento su siero e liquido
cefalorachidiano bovini: un nuovo approccio alla diagnosi delle patologie del
sistema nervoso centrale
Di Muro, G.; Tessarolo, C.; Cagnotti, G.; Favole, A.; Ferrini, S.; Bellino, C.; Borriello, G.;
Gallo, M.; Iamone, G.; Iulini, B.; Pezzolato, M.; Corona, C.; D'Angelo, A.
Oral Presentation at 2^nd Congresso multisala SIVAR-SIB
11^th - 12^th April 2024, Cremona, Italy

Attended courses and congresses
36^th ESVN-ECVN Symposium, 13^rd - 14^th September 2024, Porto, Portugal

SINVET online meeting - Il neurologo e gli occhi, 13^rd April 2024, online conference

2^nd Congresso multisala SIVAR-SIB, 11^th - 12^th April 2024, Cremona, Italy

SINVET meeting - Le patologie degenerative del sistema nervoso, 25^th - 26^th November 2023, Cremona, Italy

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