**2.5. Neuropathological consequences based on proteomic analyses**

Based on the recent analyses of SCI pathological processes, it seems that complex changes in gene and protein expression as well as in cellular interactions are taking place not only at the central lesion but also in adjacent segments. However, the exact mechanisms by which proteins involved during inflammation, recruitment and microglia activation, glial scaring, remyelination, or axonal growth function remain to be further explored [5, 10, 21, 35]. Therefore, understanding of the molecular cross-talk occurring between cells at the lesion site and in the adjacent segments needs to be further investigated [21]. In particular, studies that are able to take into account both spatial and temporal data may identify interesting molecular targets [40]. Such an investigation could be performed by a **proteomics approach**, which can be connected to cellular and physiological studies as well as to a global regeneration-activated gene (RAG) investigation. Mass spectrometry (MS) plays a central role among proteomics approaches. Several developments allow fast identification of lower abundance proteins such as cytokines and chemokines [41]. Furthermore, MS is highly used in neuroscience to discover biomarker candidates and also to study the differential expression of proteins at any given time in a proteome and they are then compared with the pattern of those from healthy ones.

IL6 and CCL20, which are known to attract T regulator lymphocytes through CCR6 binding, were expressed firstly in R1 at 3 days after SCI and secondly appeared in C1 at 7 days [40]. Furthermore, results from proteomic analysis were re-confirmed with cytokine/chemokine arrays and correlated with immunohistochemistry for neutrophils and Tregs. These experiments confirmed that neutrophils were abundantly detected in both R1 and C1 segments with a peak reached 3 days after SCI without any differences in terms of amount between each segment. However, their level decreased in time. In comparison, Tregs were present 3 days after SCI, in higher amounts in the rostral segment than in the caudal one. Their levels peak at 7 days for both segments and then decrease at 10 days [40]. These data are in line with the presence of CXCL1, CXCL3, CXCL5, CCL20, TIMP-1, and IL6 in R1 at 3 days, which are known to attract neutrophils and lymphocytes. In C1, a delay was observed in the recruitment of the Tregs, which were detected 7 days after SCI and correlated with the detection of CCL20 in C1 only at 7 days, whereas neutrophils and microglial cells were already present at 3 days [40]. Taken together, the results showed that C1 is clearly different from R1 in terms of cell types and molecular content in a time course manner, and is revealed to be a target segment for therapy. The functionality of chemokine released from injured spinal cord tissue can be evaluated by chemotaxis assay, thus investigating the BV2 (microglial) cells activation, followed by

Understanding Molecular Pathology along Injured Spinal Cord Axis: Moving Frontiers…

http://dx.doi.org/10.5772/intechopen.72118

7

Western blot, and M1/M2 polarization through CX3CR1 and CD206 expression.

expression is maintained for up to 1 month [43].

and apoptotic molecules in the caudal region.

*In vitro* chemotaxis assays confirmed that BV2 cells were highly responsive to the cytokine cocktail present in the CM from lesion and rostral sites, compared to CM from the caudal site after SCI. Interestingly, the BV2 migratory potency induced by CM derived from rostral and lesion segments was 37-fold higher compared to the ATP or LPS stimulations that increase their migration by close to 3-fold, due to the specific factors found in the complex CM [41, 42]. Furthermore, immunocytochemical studies prove that activated BV2 cells exposed to CM from the rostral segment overexpressed the CX3CR1 receptor, known to correspond with the M2 profile. This finding was strengthened by Western blot analysis and lack of labeling with C2KR, an M1 receptor [41]. These data together with *in vivo* CX3CR1 expression were in close coherence with published transcriptomic experiments showing that in the injured spinal cord, M2 gene expression is transiently expressed during 7 days after injury, while the M1 gene

Spatio-temporal proteomic analysis of spinal cord tissue between 3 and 10 days after injury provide clear evidence of regionalization between the rostral and caudal axes, with an expression of neurotrophic and immune modulatory factors in the rostral region, in contrast to inflammatory

Neutrophic factors were found at 3 and 7 days after injury and disappeared at 10 days. They were replaced by synaptogenesis factors reflecting the fact that a neurorepair process is taking place in the rostral segment after 10 days. In fact, more neurotrophic factors have been detected in the lesion and rostral parts, i.e., CTGF (connective tissue growth factor), NOV (Protein NOV homolog), PIGF (placenta growth factor), FGF-1 (fibroblast growth factor 1), BMP 2 or BMP3 (bone morphogenetic proteins (2 or 3), NGF, PGF, TGF beta (1–3) (transforming growth factor beta), periostin, GAP-43, neurotrimin, neurofascin, and hepatocyte growth factor-regulated tyrosine kinase substrate (HGS). In addition, molecules involved in neuronal development/differentiation/ neuronal migration, i.e., CRIP1 (cysteine-rich protein 1), DRP-5 (dihydropyrimidinase-related

Thus, to better understand the pathology based on secondary injury processes and plasticity, it is necessary to analyze entire spinal cord tissues in time, thus collecting tissues from the epicenter and both adjacent segments above (rostral) and below (caudal) the lesion firstly in acute, and afterwards in chronic SCI experimental models, expecting the release of different molecules. They will most likely reflect pathology *in situ*, at each specific segment, which may contribute to the final view of ascending or descending pathway disruption resulting in aggravation of clinical symptoms [41].

Nowadays, we can count on innovative proteomics technologies that can screen, identify image lipids and peptides in each spinal cord segment-derived conditioned medium (CM), or in the spinal cord tissue obtained *in vitro*, to better understand protein composition changes along the rostro-caudal axis after SCI with time in SCI.

Recently, application of shotgun proteomic analysis and label-free quantification to conditioned medium from the injured spinal cord (CM) identified chemokines (CXCL1; CXCL2; CXCL7, CCL2, CCL3, CCL22, CLCF1, and EMAP II) and neurotrophic factors (TGF, FGF-1, PDGF, and FGF1) in the lesion and rostral segments. These molecules are known to have immune-modulator and neurotrophic properties and ability to polarize macrophages/microglial cells into the M2 phenotype [10].

Chemokines are the most important molecules released immediately after SCI. Specific chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL7, CCL3, CCL20, and IL6) that are secreted by macrophages or epithelial cells after injury have the ability to attract neutrophils and lymphocytes, activate inflammation and stimulate extracellular matrix synthesis and tissue remodeling. Recent data showed that the cytokine profile changes in time between the segment above and below the lesion. This is in line with the hypothesis that immune cells that are attracted along the spinal cord upon injury insult are quite different between rostral (R1) and caudal (C1) segments in time. Recently, using proteomic analysis it has been documented that specific immune cells initially migrate toward R1 and then C1 segment [41]. In line with this, IL6 and CCL20, which are known to attract T regulator lymphocytes through CCR6 binding, were expressed firstly in R1 at 3 days after SCI and secondly appeared in C1 at 7 days [40]. Furthermore, results from proteomic analysis were re-confirmed with cytokine/chemokine arrays and correlated with immunohistochemistry for neutrophils and Tregs. These experiments confirmed that neutrophils were abundantly detected in both R1 and C1 segments with a peak reached 3 days after SCI without any differences in terms of amount between each segment. However, their level decreased in time. In comparison, Tregs were present 3 days after SCI, in higher amounts in the rostral segment than in the caudal one. Their levels peak at 7 days for both segments and then decrease at 10 days [40]. These data are in line with the presence of CXCL1, CXCL3, CXCL5, CCL20, TIMP-1, and IL6 in R1 at 3 days, which are known to attract neutrophils and lymphocytes. In C1, a delay was observed in the recruitment of the Tregs, which were detected 7 days after SCI and correlated with the detection of CCL20 in C1 only at 7 days, whereas neutrophils and microglial cells were already present at 3 days [40]. Taken together, the results showed that C1 is clearly different from R1 in terms of cell types and molecular content in a time course manner, and is revealed to be a target segment for therapy.

**2.5. Neuropathological consequences based on proteomic analyses**

aggravation of clinical symptoms [41].

6 Essentials of Spinal Cord Injury Medicine

M2 phenotype [10].

along the rostro-caudal axis after SCI with time in SCI.

Based on the recent analyses of SCI pathological processes, it seems that complex changes in gene and protein expression as well as in cellular interactions are taking place not only at the central lesion but also in adjacent segments. However, the exact mechanisms by which proteins involved during inflammation, recruitment and microglia activation, glial scaring, remyelination, or axonal growth function remain to be further explored [5, 10, 21, 35]. Therefore, understanding of the molecular cross-talk occurring between cells at the lesion site and in the adjacent segments needs to be further investigated [21]. In particular, studies that are able to take into account both spatial and temporal data may identify interesting molecular targets [40]. Such an investigation could be performed by a **proteomics approach**, which can be connected to cellular and physiological studies as well as to a global regeneration-activated gene (RAG) investigation. Mass spectrometry (MS) plays a central role among proteomics approaches. Several developments allow fast identification of lower abundance proteins such as cytokines and chemokines [41]. Furthermore, MS is highly used in neuroscience to discover biomarker candidates and also to study the differential expression of proteins at any given time in a proteome and they are then compared with the pattern of those from healthy ones. Thus, to better understand the pathology based on secondary injury processes and plasticity, it is necessary to analyze entire spinal cord tissues in time, thus collecting tissues from the epicenter and both adjacent segments above (rostral) and below (caudal) the lesion firstly in acute, and afterwards in chronic SCI experimental models, expecting the release of different molecules. They will most likely reflect pathology *in situ*, at each specific segment, which may contribute to the final view of ascending or descending pathway disruption resulting in

Nowadays, we can count on innovative proteomics technologies that can screen, identify image lipids and peptides in each spinal cord segment-derived conditioned medium (CM), or in the spinal cord tissue obtained *in vitro*, to better understand protein composition changes

Recently, application of shotgun proteomic analysis and label-free quantification to conditioned medium from the injured spinal cord (CM) identified chemokines (CXCL1; CXCL2; CXCL7, CCL2, CCL3, CCL22, CLCF1, and EMAP II) and neurotrophic factors (TGF, FGF-1, PDGF, and FGF1) in the lesion and rostral segments. These molecules are known to have immune-modulator and neurotrophic properties and ability to polarize macrophages/microglial cells into the

Chemokines are the most important molecules released immediately after SCI. Specific chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL7, CCL3, CCL20, and IL6) that are secreted by macrophages or epithelial cells after injury have the ability to attract neutrophils and lymphocytes, activate inflammation and stimulate extracellular matrix synthesis and tissue remodeling. Recent data showed that the cytokine profile changes in time between the segment above and below the lesion. This is in line with the hypothesis that immune cells that are attracted along the spinal cord upon injury insult are quite different between rostral (R1) and caudal (C1) segments in time. Recently, using proteomic analysis it has been documented that specific immune cells initially migrate toward R1 and then C1 segment [41]. In line with this, The functionality of chemokine released from injured spinal cord tissue can be evaluated by chemotaxis assay, thus investigating the BV2 (microglial) cells activation, followed by Western blot, and M1/M2 polarization through CX3CR1 and CD206 expression.

*In vitro* chemotaxis assays confirmed that BV2 cells were highly responsive to the cytokine cocktail present in the CM from lesion and rostral sites, compared to CM from the caudal site after SCI. Interestingly, the BV2 migratory potency induced by CM derived from rostral and lesion segments was 37-fold higher compared to the ATP or LPS stimulations that increase their migration by close to 3-fold, due to the specific factors found in the complex CM [41, 42]. Furthermore, immunocytochemical studies prove that activated BV2 cells exposed to CM from the rostral segment overexpressed the CX3CR1 receptor, known to correspond with the M2 profile. This finding was strengthened by Western blot analysis and lack of labeling with C2KR, an M1 receptor [41]. These data together with *in vivo* CX3CR1 expression were in close coherence with published transcriptomic experiments showing that in the injured spinal cord, M2 gene expression is transiently expressed during 7 days after injury, while the M1 gene expression is maintained for up to 1 month [43].

Spatio-temporal proteomic analysis of spinal cord tissue between 3 and 10 days after injury provide clear evidence of regionalization between the rostral and caudal axes, with an expression of neurotrophic and immune modulatory factors in the rostral region, in contrast to inflammatory and apoptotic molecules in the caudal region.

Neutrophic factors were found at 3 and 7 days after injury and disappeared at 10 days. They were replaced by synaptogenesis factors reflecting the fact that a neurorepair process is taking place in the rostral segment after 10 days. In fact, more neurotrophic factors have been detected in the lesion and rostral parts, i.e., CTGF (connective tissue growth factor), NOV (Protein NOV homolog), PIGF (placenta growth factor), FGF-1 (fibroblast growth factor 1), BMP 2 or BMP3 (bone morphogenetic proteins (2 or 3), NGF, PGF, TGF beta (1–3) (transforming growth factor beta), periostin, GAP-43, neurotrimin, neurofascin, and hepatocyte growth factor-regulated tyrosine kinase substrate (HGS). In addition, molecules involved in neuronal development/differentiation/ neuronal migration, i.e., CRIP1 (cysteine-rich protein 1), DRP-5 (dihydropyrimidinase-related protein 5), Negr1 (neuronal growth regulator 1), NCAN (neurocan core protein), CD44, Wnt8, syndecan-4, nexin, and Bcl-2, were identified. Specific factors involved in immune cell chemotaxis or cellular adhesion, including complement factors (C1qb, C1qc, factor D, factor I, and CD59), tetraspanins (CD9 and CD82), and CD14 have also been characterized [40, 41].

of decompression and reconstruction of the spine. This clearly indicates that early intervention on traumatic spinal cord injuries can significantly affect the prognosis of the disease [3]. Therefore, great attention has been paid to studies that deal with the optimal timing of surgical procedures for acute spinal cord injury [48]. Previous data suggest that patients undergoing surgical decompression within 24 h after spinal cord injury have a significantly better recovery prognosis [29]. Currently, a number of innovative neuroprotective strategies for acute spinal cord injuries are being tested and evaluated in randomized controlled trials. Experimental studies on animal models showing promising results, such as ChABC, minocycline, riluzole, granulocyte colony stimulating factor (G-CSF), are now being tested in clinical studies [2, 49]. Hypothermia induced by intravascular cooling infusion administered epidural or subcutane-

Understanding Molecular Pathology along Injured Spinal Cord Axis: Moving Frontiers…

http://dx.doi.org/10.5772/intechopen.72118

9

Pharmacotherapy is one of the most widespread forms of treating secondary damage that use a wide variety of different types of molecules to target specific secondary processes. These are comprised of anti-inflammatory or neurostimulating compounds such as, minocycline, neurotrophic factors (BDNF, GDNF, NGF, and erythropoietin), and molecules that alleviate

In particular, chondroitinase ABC eliminate CSPG with the major component NG2 which inhibits the regeneration of damaged axons [50]. Nogo-neutralizing antibodies or blockers of the post-receptors components RhoA, are used to improve long-distance axon regeneration and sprouting [25]. Previous studies have identified Rho pathway as important to control the neuronal response after CNS injury. Therefore, a drug called Cethrin® that blocks activation of Rho is actually in phase I/IIa of clinical trials [48]. The most encouraging findings were observed in patients with cervical SCI, whereas patients with injuries at thoracic level received only modest neurological recovery. Although the patient numbers were small in this trial, the results obtained indicate some evidence of efficacy to enhance functional recovery and warrant further

regenerating axons from the inhibitory effects of extracellular matrix molecules.

**3.2. Molecular therapies: chondroitinase ABC, minocycline, tacrolimus, riluzole**

**Chondroitinase ABC** is a bacterial enzyme that reduces the inhibitory effect of CSPGs at the site of injury. In order to increase CNS regeneration, only chondroitinase ABC purified from Proteus vulgaris [52] should be delivered. The mechanism of action lies in removing GAG chains from the nuclear protein and converting them to unsaturated disaccharides [34]. These stimulate the release of growth factors and proteins attached to GAGs of CSPGs, thereby enabling their diffusion and interaction with neural cell receptors. ChABC has been shown to promote neuroprotection and neuroregeneration [53]. Experimental administration of ChABC after cervical SCI positively affects the branching of damaged and intact descending pathways around which increased accumulation of CSPGs and then inhibition of axonal growth occurs. The neuroprotective effect of ChABC has been described also for the hemisection of the spinal cord [50, 54], transection of dorsal columns [55], and after compression injury of the thoracic spinal cord and the peripheral nerve [56] or in adult rats with visual deficits [57].ChABC administration is often

ously has achieved success during acute SCI treatment.

**3.1. Pharmacotherapy**

clinical trials [51].

In contrast, proteins produced in the caudal region were related to necrosis factors (BAX, BAD, Caspase 6, and neogenin), cytoskeleton proteins, synaptic vesicle exocytosis, chemoattractant factors, and neuronal postsynaptic density.

These data are in line with our previous *in vivo* results demonstrating that neurite outgrowth takes place from rostral to lesion but never in the opposite direction from caudal to the lesion. Furthermore, the presence of chemokines, lectins, and growth factors in the rostral but not in the caudal segment clearly document the immediate inflammatory response together with activity-dependent factors released by neurons and glia.

In order to investigate the neurotrophic role of CM derived from the injured tissue, studies testing neurite outgrowth in rat DRG explants have been undertaken. Data from these experiments confirmed that enhanced neurite sprouting of DRGs facilitated by CM from rostral and lesion segments were most likely mediated by the content of neurotrophic factors, i.e., FGF-1, NGF, PGF, BMP 2 or BMP3, GAP-43, neurotrimin, neurofascin, and other molecules involved in neuronal development/differentiation/migration. Although the principal role of NGF/TrkA pathways in sensory axon outgrowth has been widely demonstrated, other neurotrophic factors including the BMPs (members of the TGFβ superfamily) or GAP-43 have to be taken into account [41, 44].

In summary, it has been demonstrated that few days after SCI, a clear regionalization occurs between the rostral and caudal axes, with expression of neurotrophic and immunomodulatory factors in the rostral region, in contrast to inflammatory and apoptotic molecules in the caudal region. These data indicate the importance of stimulating neurite sprouting at segments below the lesion by inhibiting inflammation and turning polarization of M1 cells to the M2 state, which could have a clear impact on neurorepair. Therefore, these findings should be taken into account when planning new treatment strategies.
