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EVALUATION OF PATHOGENESIS OUTCOMES
OF INTERVERTEBRAL DISC TISSUE IN PATIENTS WITH HERNIATED DISC BASED ON HISTOPATHOLOGICAL AND HISTOLOGICAL GRADING
D.M. Al-Muathen1, S.A.H. Al-Sharqil, S.A. Brakhas2, A.A. Taker3
1 Department of Biology, College of Science, Mustansiriyah University, Baghdad, Iraq;
2 Department of Immunology, Allergy Specialized Center, Ministry of Health, Baghdad, Iraq;
3 Ghazy Al-Hariri Hospital for surgical specialties, Medical City Complex, Baghdad, Iraq.
* Corresponding author: [email protected]
Abstract. Background: the nucleus pulposus NP pulls on the ruptured annulus fibrosus AF, bulging the intervertebral disc IVD and releasing chemicals that may irritate nerves and cause inflammation and pain. This produces histological changes to the IVD, including less gelatinous NP, cracks and fissures, decreased matrix water content, and proteoglycan composition changes. Aim of study: this study aims to investigate the histopathological changes in the Herniated Disc HD tissue, as well as cartilage histopathology grade and stage assessment. Materials and methods: fourty tissue samples of lumbar HD obtained after the operations were kept in 10% formalin. Then all HD sections were processed, and embedded, after that, 5 ^m thick glass mounted sections were stained with Hematoxylin and Eosin and Alcian blue (pH 0.02) and examined microscopically for histopathological changes and grading scoring was also conducted based on cell density, structural alterations of collagen fibers, and proteoglycan degeneration. Results: hemorrhage with fibrin deposition, mucous degeneration around chondrocyte clones and degeneration, increased chondrocyte density, expanded lacunae with degenerated chondrocytes, fiber disorientation, cleft formation, mucoid matrix changes, and inflammatory cell infiltration with fibrocyte prefiltration were the most histopathological changes in HD samples. Along with necrotic chondrocyte increase and tissue fiber alterations. Histological cell density grade indicated different-sized clones. All HD tissues revealed collagen fiber structural alterations, with gradients (52.5%) being most common. All HD samples showed mucous (proteoglycans) degradation, especially in gradients abundantly present and intermediate between 1 and 3 (45% and 42.5%). Conclusions: histopathological changes in intervertebral disc associated with HD infection.
Keywords: lumbar spine, herniated disc, histopathological study, grading score.
List of Abbreviations
AF - Annulus fibrosus
CEPs - Cartilaginous end plates
ECM - Extracellular matrix
HD - Herniated Disc
IDD - Intervertebral disc degeneration
IVD - Intervertebral disc
MMPs - Matrix metalloproteinase
NP - Nucleus pulposus
Introduction
Millions of individuals throughout their lives deal with low back pain (LBP), making it one of the most prevalent chronic conditions (Froud et al., 2014). The degeneration of the intervertebral discs (IVDs) is one of the potential causes of this disorder (Dowdell et al., 2017). However, there is a great deal of individual variation in the clinical presentation of varying
degrees of IVD degeneration (Siemionow et al., 2011). It is possible for someone to have significant radicular symptoms in their legs while experiencing mild, moderate, or nonexistent low back pain (Bardin et al., 2017). It is also not entirely known how IVD degeneration relates to the patients' clinical presentation (Whatley & Wen, 2012). Similar to the aging impact on the IVD, degenerative disc degeneration is characterized by a diffusion delay over the endplates (Rajasekaran et al., 2004). Herniated lumbar discs are the leading cause of radicular and sciatica pain. This disease has a varied prevalence ranging from 1.6% to 43% and a lifetime prevalence of roughly 10%. One of the leading causes of occupational incapacity is this kind of discomfort, which radiates downward down the lower limbs (Kumar et al., 2011). While there has been a lot of research on the radiographic
and histological features of IVD herniation and degeneration, the correct association with patient symptoms has not been determined just yet (Khan et al, 2017). In clinical practice, it is not uncommon to see cases with IVD degeneration or herniation when symptoms are mild or nonexistent (Kepler et al., 2013). In addition, calcification is one possible consistency of a herni-ated disc, which may lead to more complex surgical procedures and subsequent problems (Ruetten et al., 2018). Upon macroscopic inspection, the IVD is composed of an outer layer of fibrous cartilage called the AF and an inner layer of gelatinous material called the NP. Cartilaginous end plates CEPs border the NP on each side, coating it inferiorly and superiorly (Huang et al., 2018). The proteoglycans consist 65% of the dry weight of the nucleus. And the matrix of the nucleus refers to the mixture of aggregates, collagen fibrils, and proteoglycan units found inside the NP, which also includes a small number of chondrocyte-like cells (To-maszewski et al, 2015). Collagen accounts for 50-60% of the dry weight of the AF, whereas proteoglycans make up the remaining 20%. Separate lamellae filled with a proteoglycan gel, formed by chondrocytes (which are found deeper in the annulus, towards the nucleus) and fibroblasts (which are positioned mostly towards the periphery of the annulus) lie between the collagen fibers (Williams et al, 2019). Also, the CEPs contain Proteoglycans, and type II collagen fibers that run horizontally and parallel to the vertebral bodies and make up the bulk of the disc, with the fibers extending into the disc. Cartilage cells line up neatly in a row along the collagen strands (Guerrero et al, 2021).
A tear in the outer fibrous ring AF of an IVD, allowing the soft center section NP to protrude beyond the disc, is an anatomical anomaly known as lumbar HD (Yu et al.,2022). In the HD, many histological alterations occurred, as the nucleus became less gelatinous fractures and fissures formed over time, matrix water content decreased and proteoglycan composition changed throughout time (Amin et al, 2017). This is followed by a shift in collagen synthesis, with decreased production of type II
collagen and increased production of type I and type III collagen (Singh et al.,2023). An increase in cell death (Apoptotic NP cells) is accompanied by an increase in cell proliferation and the creation of cell clusters (Maher et al, 2017). Based on their examination of lumbar specimens from cadavers spanning from infancy to old age, Lai et al. (2021) categorised the histological alterations in the deteriorating IVDs. According to their research, the most prevalent alterations in degenerative IVDs are rim lesions and edge neovascularization, chondrocyte proliferation, lesions or cleft development, mucoid degeneration, and granular changes, they suggested a comprehensive system for rating the histological alterations in the deteriorated lumbar IVDs (Tomaszewski et al., 2017).
Materials and Methods
Collection of the herniated disc tissue specimens
Fourty individuals were surgically diagnosed with HD, and tissue samples were stored in the fixative solution (formalin 10%) immediately after they were taken during endoscopic discectomy procedures (Fig. 1) at Ghazy Al-Hariri Hospital for Surgical Specialties/Medical City complex and Al-Zaytoon Hospital for general surgery (private), from February 2022 to December 2022.
Histological technique
After fixation, all HD samples were processed, embedded in paraffin, and 5-micron thick glass mounted sections were routinely stained with Hematoxylin and Eosin and Alcian blue (pH 0.02) stains for preparing for histo-pathological study according to the Suvarna et al. (2019) method.
Histopathological grading and staging of HD tissue
Total of 40 patients with HD, the tissue specimens were preoperatively by using Weiler et al. (2011) for grading and staging method, and modified parameters were collected as follows:
1. Cell density (chondrocyte)
0 = no proliferation, 1 = increased cell density 2 = connection of two chondrocytes 3 =
Fig. 1. The herniated disc specimens after the endoscopic discectomy operation
= small-size clones (several chondrocytes grouped, 3-7 cells) 4 = moderate size clones (8-15 cells), 5 = huge clones (> 15 cells).
2. Structural alterations of collagen fibers
0 = absent, 1 = rarely present 2 = present in intermediate amounts between 1 and 3 3 = = abundantly present, 4 = scar/tissue defects
3. Proteoglycans degeneration
0 = absent, 1 = rarely present 2 = present in intermediate amounts between 1 and 3, 3 = = abundantly present
All stained HD sections were examined under a light microscope and photographed the selected fields that show our results.
Ethical approval
All participants agreed to provide the investigator with the specimens. The ethics committee of the College of Science, Mustansiriyah University approved this work. Informed consent according to the Declaration of Helsinki was obtained from all participants.
Results
Histopathological study of the intervertebral disc of HD patients
Fourty herniated disc tissues were obtained from the lumbar prolapsed patients. After
staining with H&E and Alsian blue stains, light microscopy revealed histological changes. These changes are:
1 - Hemorrhage with deposition of fibrin in many areas as shown in Figure 2a. And increase in chondrocyte density in all sections as shown in Figure 2b.
2 - Moreover, the results showed mucous degeneration areas around clones of chondro-cytes as well as tissue fibers and chondrocytes appeared disorientation as shown in Figure 3.
3 - However, the results of this study showed that expanded lacunae contain degenerated chondrocytes as revealed in Figure 4a, and tissue fibers and chondrocytes appeared disorientation as shown in Figure 4b.
4 - Also, the results showed that, the formation of clefts and mucoid matrix changes as shown in Figure 5a. Moreover, the results of this study show inflammatory cell infiltration with prefiltration of fibrocytes as shown in Figure 5b.
5 - Furthermore, in this study, the results showed the accumulation number of necrotic chondrocytes with structure tissue fibers alterations as shown in Figure 6a, and accumulation number of dead cells with irregular network fibers as shown in Figure 6b.
Fig. 2. Section in intervertebral disc tissue of HD patient showed: A) hemorrhage (head arrows) with deposition of fibrin in many areas (black arrows); B) increase of chondrocytes density (arrows) (Hematoxylin and Eosin staining, large figure X4, small figure X10)
Fig. 3. Section in intervertebral disc tissue of HD patient showed mucous degeneration around clones of chondrocytes (red arrows) with disorientation of fibers (double arrow) (A - Hematoxylin and Eosin staining, B - Alcian blue stain, X10)
Fig. 4. Section in intervertebral disc tissue of HD patient showed: A) expanded lacunae contain degenerated chondrocytes (arrows); B) tissue fibers (red arrows) and chondrocytes (black arrows) appeared disorientation (Hematoxylin and Eosin staining, large figure X4, small figure X10)
Fig. 5. Section in intervertebral disc tissue of HD patient showed: A) formation of clefts (arrows) and mucoid matrix changes; B) inflammatory cells infiltration (red arrow) with prefiltration of fibrocytes (black arrows) (Hematoxylin and Eosin staining, X10)
Fig. 6. Section in intervertebral disc tissue of HD patient showed: A) accumulation number of necrotic chondrocytes (red arrow) with structure tissue fibers alterations; B) accumulation number of dead cells (red arrows) with irregular network fibers (Alcian blue staining, X10)
Histological grading and staging of HD patients
In addition, microscopic examination of HD tissues from patients investigated in Table 1, revealed (according to cell density grade) no proliferation in chondrocytes in 2.5% of the specimens. While the highest percentage (27.5%) in small-size clones (several chondrocytes grouped, 3-7 cells) gradient, compared to hug clones (>15 cells) gradient (22.5%). On the other hand, the connection of two chondrocytes gradient (20%), increased cell density gradient (15%), and moderate size clones (8-15cells) (12.5%). Also, the results
showed structural alterations of collagen fibers in all studied HD tissues, and that are found (at 52.5%) in abundantly present gradient, (22.5%) in present in intermediate amounts between (1 and 3) gradient, (17.5%) in scar/tissue defects gradient (Figure 7a and b), and finally (7.5%) in rarely present gradient (Table 1). Moreover, the present results revealed mucous (proteoglycans) degeneration in all HD tissues sections, and that are recorded (45%) in abundantly present gradient, (42.5%) in present in intermediate amounts between 1-3 gradient, and (12.5%) in rarely present gradient (Table 1).
Table 1
Histological grading and staging of intervertebral disc tissue of HD patients
Criteria / Grading HD patients NO HD patients (%)
1. Cell density (chondrocyte) - -
0 = no proliferation 1 2.5
1 = increased cell density 6 15
2 = connection of two chondrocytes 8 20
3 = small size clones (several chondrocytes grouped, 3-7 cells) 11 27.5
4 = moderate size clones (8-15 cells) 5 12.5
5 = huge clones (> 15 cells) 9 22.5
Total 40 100
2. Structural alterations of collagen fibers - -
0 = absent 0 0
1 = rarely present 3 7.5
2 = present in intermediate amounts between 1 and 33 = abun- 9 22.5
dantly present 21 52.5
4 = scar/tissue defects 7 17.5
Total 40 100
3. Mucous (Proteoglycans) degeneration - -
0 = absent 0 0
1 = rarely present 5 12.5
2 = present in intermediate amounts between 1 and 3,3 = abun- 17 42.5
dantly present 18 45
Total 40 100
Fig. 7. Section in intervertebral disc tissue of HD patient showed: A) scar formation with mucous degeneration around clone; B) mucous degeneration around clone (Alcian blue staining, X10)
Discussion
Microscopically, the sections of IVD tissue of HD patients using Hematoxylin and Eosin and Alcian blue (pH 0.02) stains showed hemorrhage with deposition of fibrin in many areas. In degenerative disc disease, the IVDs gradually deteriorate over time. They may be-
come more fragile and susceptible to small tears or injuries. These tears can cause bleeding and the deposition of fibrin as the body attempts to repair the damaged tissue. And in some cases, infections may cause damage to blood vessels and surrounding tissues, while inflammation can trigger a cascade of immune responses, in-
eluding fibrin formation (Desmoulin et al., 2020). On the other hand, in all sections, it was demonstrated an increase in chondrocyte density that may be related to the body's tries to restore or compensate for the damaged tissue. Furthermore, HD can lead to tissue damage and inflammation, prompting chondrocytes to proliferate in an attempt to repair the damaged area (Xiao et al., 2021). In addition, the results showed mucous degeneration areas around clones of chondrocytes as well as tissue fibers and chondrocytes appeared disorientation. These histopathological observations can be indicative of a specific condition known as "mucinous degeneration" of the IVD primarily affecting the NP and it is characterized by the accumulation of gelatinous material within the disc tissue, and also around the clusters of chondrocytes (Murphy et al., 2023). Moreover, In degenerative disc disease, there is often a loss of ECM integrity within the disc, which includes collagen fibers and proteoglycans. This loss of integrity can result in a disorganized arrangement of these structural elements, affecting the overall architecture of the disc (Kirnaz et al., 2022). However, the results of this study showed that expanded lacunae contain degenerated chondrocytes, and tissue fibers and chon-drocytes appeared disorientation. When the disc experiences degenerative changes, such as those associated with aging or injury, chondrocytes could undergo degeneration and cell death (Feng et al, 2022). On the other hand, Krut et al. (2021) demonstrated that, as individuals age, the IVDs naturally undergo degenerative changes. These changes could involve the progressive loss of viable chondrocytes and alterations in the extracellular matrix ECM composition, leading to the expansion of lacunae containing degenerated chondrocytes. Furthermore, the results showed that, the formation of clefts and mucoid matrix changes. Intervertebral disc degeneration IDD often leads to the formation of clefts or fissures within the disc. These clefts represent areas where the disc's structural integrity has been compromised due to the breakdown of collagen fibers and proteo-glycans, and in this condition, the disc undergoes structural and compositional changes over
time (Scarcia et al., 2022). Moreover, the results of this study show inflammatory cell infiltration with prefiltration of fibrocytes. Inflammation can be a response to the degenerative changes in the tissue and may involve immune cells like macrophages and neutrophils. These cells can release inflammatory mediators and enzymes that contribute to tissue damage (Ye et al, 2022). Abdul Sahib et al. (2017) revealed infiltrated macrophages and lymphocytes and proliferation of fibrocystic in degenerated posterior cruciate ligament obtained from the knee osteoarthritis. The prefiltration of fibrocytes refers to the presence of fibroblast-like cells within the IVD, these cells may migrate into the disc tissue as part of the body's attempt to repair or adapt to the degenerative changes. Fibrocytes can be involved in the production of ECM components, but their activity may be disorganized in the context of degeneration (Theo-charis et al., 2019). Also, in this study, the results showed that, the accumulation number of necrotic chondrocytes with structure tissue fibers alterations, and accumulation number of dead cells with irregular network fibers. Cell death, a functional biological process required for cellular development, is classified as apop-tosis, necrosis, or autophagy. Cell death modulation is associated with various diseases and highly contributes to IDD. It was characterized by the progressive breakdown of the disc's structure and function (Zhang et al, 2021). The IVD was avascular, meaning it lacked a direct blood supply. It relies on diffusion from nearby blood vessels for nutrients and oxygen. In advanced stages of degeneration, necrosis refers to the death of chondrocytes within the IVD. Accumulation of necrotic chondrocytes was a sign of severe tissue damage, as the disc degenerates, this diffusion becomes less efficient, leading to reduce blood supply and nutrition to the cells which reduces their viability and then causes necrosis (Wang et al., 2016).
Furthermore, microscopic examination of HD tissues from patients was revealed according to cell density grade that no proliferation in chondrocytes in (2.5%) of the specimens. While the highest percentage (27.5%) in small-size clones (several chondrocytes grouped, 3-7
cells) gradient, compared to hug clones (>15 cells) gradient (22.5%). Also the connection of two chondrocytes gradient (20%), increased cell density gradient (15%), and moderate size clones (8-15cells) (12.5%). In addition, the results showed structural alterations of collagen fibers in all studied HD tissues. And that are found (at 52.5%) in abundantly present gradient, (22.5%) in present in intermediate amounts between (1 and 3) gradient, (17.5%) in scar/tissue defects gradient, and finally (7.5%) in rarely present gradient. On the other hand, the present results revealed mucous (proteogly-cans) degeneration in all HD tissues sections, and that are recorded (45%) in abundantly present gradient, (42.5%) in present in intermediate amounts between 1-3 gradient, and (12.5%) in rarely present gradient. In response to IDD and mechanical stress, chondrocytes within the disc may become more active. This increased activity can lead to alterations in tissue structure and organization. Chondrocytes may attempt to repair damaged areas or adapt to changes in the microenvironment by producing matrix components, but this response may be disorganized (Das et al., 2021). Moreover, in IDD the IVD undergoes wear and tear due to repeated mechanical stresses. This could lead to the breakdown of the ECM, which was primarily composed of collagen and proteoglycans (Stokes & Iatridis, 2004). Chondrocyte cells were responsible for maintaining this matrix. As the IVD ages, the chondrocytes may start to divide and form clones in an attempt to repair the damaged tissue. Cloning can occur as a response to tissue damage or as an effort to maintain the disc's structural integrity (Yurube et al, 2012). Also study by Morris et al. (2017) found that when the IVD degenerates the ECM begins to degrade, and the body's natural response is to recruit more cells to the area to try to repair it, and the increased cell density could also be a part of the body's inflammatory response to injury. These additional cells, including chondrocytes and inflammatory cells, could contribute to the formation of clones as they attempt to address
the tissue damage. In addition, Cauble et al. (2020) found the ECM's Collagen fibers may become disorganized or fragmented, and prote-oglycan content may decrease in severe degenerative disc disease, and these alterations contribute to the disc's loss of structural integrity. Furthermore, traumatic injuries or repetitive mechanical stress on the IVD could lead to damage to the collagen fibers. This damage triggers a repair response, leading to the deposition of scar tissue. Scar tissue often consists of collagen fibers that are irregularly arranged and may not function as effectively as the original healthy collagen network (Tang et al, 2022). An atrophy and degeneration of the mucous proteoglycan substance in the histopatho-logical section of the IVD were characteristic features often associated with IDD and aging, as individuals age, the IVDs undergo degenerative changes. These changes could include a decrease in the proteoglycan content of the disc's NP (Lee et al, 2022). In addition, in IDD or injury, the Inflammatory mediators can stimulate the release of enzymes like matrix metallopro-teinase (MMPs) that degrade proteoglycans and other extracellular matrix components (Dolma & Kumar, 2021).
Conclusions
Results from our present study demonstrate that histopathological changes in intervertebral discs are associated with HD infection. As intervertebral disc cells tend to cluster in clones, structural alterations of collagen fibers and pro-teoglycan degeneration were most common.
Acknowledgment
The authors would like to introduce their thanks and gratefulness to Mustansiriyah University (https://www.uomustansiriyah.edu.iq/) for the support and help to achieve this article. And I should express my gratitude to Dr. Khalid Muhssin Murad MSc. Orthopedic Surgery, Al-Zaytoon Hospital, chief of the Iraqi Pain Society, for his assistance and for facilitating my clinical part of the study.
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