Effect of applying p75NTR saporin to a punctured intervertebral disc on calcitonin gene-related peptide expression in rat dorsal root ganglion neurons

Background

Recent studies have revealed that the low-affinity nerve growth factor receptor, p75 neurotrophin receptor (p75NTR), is important in inflammatory pain. Moreover, p75NTR immunoreactive sensory nerve and dorsal root ganglion (DRG) neurons have been found to innervate lumbar intervertebral discs. The purpose of the current study was to investigate the effect of p75NTR saporin, a toxin used to destroy p75NTR, on calcitonin gene-related peptide (CGRP), an inflammatory neuropeptide associated with pain, in DRG neurons innervating punctured intervertebral discs in rats.

Methods

The neurotracer fluorogold (FG) was applied to the surfaces of L5/6 discs to label their innervating DRG neurons (n = 30). Of 30 rats, 10 were in a nonpunctured disc sham surgery control group (nonpuncture group), and the other 20 were in experimental groups in which intervertebral discs were punctured with a 23-gauge needle. p75NTR saporin was applied to the discs of 10 rats (puncture + p75NTR saporin group) and the other 10 received the same volume of saline (puncture + saline group). At 14 days after surgery, DRGs from L1 to L6 were harvested, sectioned, and immunostained for CGRP, and the proportions of CGRP-immunoreactive DRG neurons was evaluated.

Results

Of the FG-labeled neurons innervating the L5/6 disc, the proportion of CGRP-immunoreactive neurons was 32% ± 6% (mean ± SE) in the nonpuncture group, 47.2% ± 8% in the puncture + saline group, and 34.6% ± 9% in the puncture + p75NTR saporin group. The proportion of CGRP-immunoreactive neurons was significantly greater in the puncture + saline group compared with the nonpuncture and puncture + p75NTR saporin groups (P < 0.01).

Conclusions

Half of the DRG neurons innervating the discs were positive for CGRP in the puncture + saline group. CGRP is important for mediating inflammatory and nerve-injured pain and may be important in discogenic pain. However, p75NTR saporin suppressed CGRP expression in DRG neurons. Therefore, p75NTR may be an important receptor for mediating discogenic pain via CGRP expression.     

Intervertebral discs which cause low back pain secrete high levels of proinflammatory mediators

Herniated intervertebral disc tissue has been shown to produce a number of proinflammatory mediators and cytokines, but there have been no similar studies using discs from patients with discogenic low back pain. We have compared the levels of production of interleukin-6 (IL-6), interleukin-8 (IL-8) and prostaglandin E2 (PGE2) in disc tissue from patients undergoing discectomy for sciatica (63) with that from patients undergoing fusion for discogenic low back pain (20) using an enzyme-linked immunoabsorbent assay. There was a statistically significant difference between levels of production of IL-6 and IL-8 in the sciatica and low back pain groups (p < 0.006 and p < 0.003, respectively). The high levels of proinflammatory mediator found in disc tissue from patients undergoing fusion suggest that production of proinflammatory mediators within the nucleus pulposus may be a major factor in the genesis of a painful lumbar disc.     

Changes in water content of intervertebral discs and paravertebral muscles before and after bed rest

Background  Although low back pain can be principally produced or increased during action, it may also be induced or enhanced in the morning after bed rest. During bed rest, tissue edema (increased water content) may occur. In this study, we measured the changes in water content in the intervertebral disc and the paravertebral muscle before and after bed rest using a magnetization transfer magnetic resonance imaging (MT-MRI) technique that permits measuring water content in tissues. Methods  A total of 20 student volunteers were enrolled in this study. MT-MRI evaluation was performed before and after bed rest. To measure water content in the intervertebral disc and paravertebral muscle, two MRI sequences were performed using MT pulse-off and MT pulse-on. Based on the two images obtained, the equivalent cross-relaxation rate (ECR) was calculated. Results  The ECR for intervertebral discs was significantly lower after bed rest than before bed rest (P < 0.01). The ECR for paravertebral equivalent cross-relaxation rate muscles was significantly higher after bed rest than before bed rest (P < 0.05). Conclusion  We obtained results indicating that after bed rest the water content in the intervertebral disc and the paravertebral muscle was increased and decreased, respectively.     

Existence of brain-derived neurotrophic factor and vanilloid receptor subtype 1 immunoreactive sensory DRG neurons innervating L5/6 intervertebral discs in rats

 The rat L5/6 intervertebral disc is innervated by L1 to L6 dorsal root ganglia (DRGs). T13 to L2 DRGs innervate the L5/6 intervertebral disc through paravertebral sympathetic trunks, whereas L3 to L6 DRGs directly innervate through sinuvertebral nerves on the posterior longitudinal ligament. The presence of substance P (SP)-immunoreactive (ir) and calcitonin gene-related peptide (CGRP-ir) sensory nerve fibers on the lumbar intervertebral disc has been established. SP and CGRP are markers of sensory neurons mainly involved with pain perception. The existence of SP-ir and CGRP-ir DRG neurons innervating the L5/6 intervertebral disc has been also demonstrated. Brain-derived neurotrophic factor (BDNF), which exists mainly in the small DRG neurons, plays an important neuromodulatory role in inflammatory conditions. Vanilloid receptor subtype 1 (VR1) in the DRG neurons and spinal dorsal horn is a channel that appears to confer responsiveness to heat and chemical stimuli. The presence of BDNF-ir and the VR1-ir DRG neurons innervating the L5/6 intervertebral disc has not. In this study of DRG neurons innervating the L5/6 intervertebral disc, the proportions of BDNF-ir in L1, L2, L3, L4, and L5 DRG neurons were 14%, 12%, 12%, 12%, and 13% and the proportions of VR1-ir L1, L2, L3, L4, and L5 DRG neurons were 10%, 8%, 24%, 19%, and 23%, respectively. Under physiological conditions in rats these neurons may transmit inflammatory and burning pain of the L5/6 intervertebral disc. Received: March 6, 2002 / Accepted: August 12, 2002 Acknowledgments. We thank Dr. Makoto Tominaga (Mie University) for providing the anti-VR1 antiserum and Miss Keiko Kitajo for technical assistance. Offprint requests to: S. Ohtori, Anesthesiology Research, 0629, 9500 Gilman Drive, La Jolla, CA 92093-0629, USA     

Development of an in vitro intervertebral disc innervation model to screen neuroinhibitory biomaterials

Pain originating from an intervertebral disc (discogenic pain) is a major source of chronic low back pain. Pathological innervation of the disc by pain-sensing nerve fibers is thought to be a key component of discogenic pain, so treatment with biomaterials that have the ability to inhibit neurite growth will greatly benefit novel disc therapeutics. Currently, disc therapeutic biomaterials are rarely screened for their ability to modulate nerve growth, mainly due to a lack of models to screen neuromodulation. To address this deficit, our lab has engineered a three dimensional in vitro disc innervation model that mimics the interface between primary sensory nerves and the intervertebral disc. Further, herein we have demonstrated the utility of this model to screen the efficacy of chondroitin sulfate biomaterials to inhibit nerve fiber invasion into the model disc. Biomaterials containing chondroitin-4-sulfate (CS-A) decrease neurite growth in a uniform gel and at an interface between a growth-permissive and a growth-inhibitory gel, while chondroitin-6-sulfate (CS-C) is less neuroinhibitory. This in vitro model holds great potential for screening inhibitors of nerve fiber growth to further improve intervertebral disc replacements and therapeutics. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1016-1026, 2020     

Disc degeneration of cervical spine on MRI in patients with lumbar disc herniation: comparison study with asymptomatic volunteers

An association between progression of cervical disc degeneration and that of lumbar disc degeneration has been considered to exist. To date, however, this association has not yet been adequately studied. Age-related changes in the cervical intervertebral discs were evaluated by magnetic resonance imaging (MRI) in patients with lumbar disc herniation, and compared with the MRI findings of healthy volunteers without lower back pain. The purpose of this study was to clarify whether the prevalence of asymptomatic cervical disc degeneration is higher in patients with lumbar disc herniation than in healthy volunteers. The study was conducted on 51 patients who were diagnosed as having lumbar disc herniation and underwent cervical spine MRI. The patients consisted of 34 males and 17 females ranging in age from 21–83 years (mean 46.9 ± 14.5 years) at the time of the study. The control group was composed of 113 healthy volunteers (70 males and 43 females) aged 24–77 years (mean 48.9 ± 14.7 years), without neck pain or low back pain. The percentage of subjects with degenerative changes in the cervical discs was 98.0% in the lumbar disc herniation group and 88.5% in the control group (p = 0.034). The presence of lumbar disc herniation was associated significantly with decrease in signal intensity of intervertebral disc and posterior disc protrusion in the cervical spine. None of the MRI findings was significantly associated with the gender, smoking, sports activities, or BMI. As compared to healthy volunteers, patients with lumbar disc herniation showed a higher prevalence of decrease in signal intensity of intervertebral disc and posterior disc protrusion on MRI of the cervical spine. The result of this study suggests that disc degeneration appears to be a systemic phenomenon.     

神经营养素在椎间盘源性腰痛机制中的作用

椎间盘源性腰痛是慢性腰痛常见的类型,然而,其疼痛机制尚不明确。越来越多的证据表明,神经营养素在椎间盘源性腰痛机制中具有重要地位。本文从神经营养素在椎间盘、脊根神经节和脊髓后角等疼痛传递通路中的作用,对其在椎间盘源性腰痛机制中的研究进展进行综述。通过调控神经营养素及其受体来改变疼痛信息传递,为椎间盘源性腰痛的治疗提供新思路。     

Percutaneous posterior-lateral lumbar interbody fusion for degenerative disc disease using a B-Twin expandable spinal spacer

Degenerative disc disease (DDD) causes gradual intervertebral space collapse, concurrent discogenic or facet-induced pain, and possible compression radiculopathy. A new minimal invasion procedure of percutaneous posterior-lateral lumbar interbody fusion (PPLIF) using a B-Twin stand-alone expandable spinal spacer (ESS) was designed to treat this disease and evaluated by follow-up more than 1 year. 12 cases with chronic low back pain and compressive radiculopathy due to DDD refractory were selected to conservative treatment. Under fluoroscopy in the posterior-lateral position, a K-wire was advanced into the intervertebral space and a dilator and working cannula were introduced into the disc space step by step. Discectomy and endplate scratching were performed through the cannula using pituitary forceps and endplate curettage. An ESS was inserted into the intervertebral space by a B-Twin expandable spinal delivery system after some bone graft chips implanted into the disc space. The ongoing study includes intraoperative difficulties, complications, radiologic evidence of fusion and clinical outcome as scored by pre- and postoperative questionnaires pertaining to pain intensity and degree of disability. The 12 procedures of lumbar interbody fusion using stand-alone expandable spinal system through percutaneous approach were successful. Radiologic study demonstrated fusion in a total of 11 cases and only 1 exception after more than 1 year visiting. The values of Visual Analog Scale (VAS) on movement and Oswestry Disability Index (ODI) dropped by more than 80 and 67.4%, respectively. Disk space heights averaging 9.0 mm before procedure were increased to 11.5 mm 1 month (a significant difference compared with preprocedure, P < 0.01) after surgery and stabilized at 10.8 mm upon final follow-up (a significant difference compared with preprocedure, P < 0.01). The results demonstrated that the percutaneous approach for posterior-lateral lumbar interbody fusion using expandable spinal system is a valuable micro-invasion method for the DDD patients and can achieve the same outcome as with other methods.     

Diagnostic criteria and treatment of discogenic pain: a systematic review of recent clinical literature

Background contextPain innate to intervertebral disc, often referred to as discogenic pain, is suspected by some authors to be the major source of chronic low back and neck pain. Current management of suspected discogenic pain lacks standardized diagnosis, treatment, and terminology.PurposeIn an attempt to determine whether patterns existed that may facilitate standardization of care, we sought to analyze the terminologies used and the various modes of diagnosis and treatment of suspected discogenic pain.Study designA systematic review of the recent literature.MethodsA Medline search was performed using the terms degenerative disc disease, discogenic pain, internal disc disruption while using the limits of human studies, English language, and clinical trials, for the last 10 years. The search led to a total of 149 distinct citations, of which 53 articles, where the intervertebral disc itself was considered the principal source of patient's pain and was the main target of the treatment, were retained for further analysis.ResultsThe results of this review confirm and help quantify the significant differences that existed in the terminology and all the areas of diagnosis and treatment of presumed discogenic pain.ConclusionsOur findings show that suspected discogenic pain, despite its extensive affirmation in the literature and enormous resources regularly devoted to it, currently lacks clear diagnostic criteria and uniform treatment or terminology.     

Diagnosis of discogenic low back pain in patients with probable symptoms but negative discography

Objective  

The purpose of the current study was to determine, whenever the patients complained of probable symptoms of discogenic low back pain and had obvious disc pathological changes on magnetic resonance imaging (MRI) but showed negative in discography, whether we could absolutely exclude the diagnosis of discogenic pain or not.     

Discography and discogenic pain

Disc abnormalities, either directly or indirectly, are responsible for almost half the cases of back pain. A degenerated intervertebral disc without any impingement on adjacent nerve roots was suspected as a source of low-back pain in the 1920s. Discography is now considered a diagnostic procedure designed to determine whether a disc is intrinsically painful. The procedure involves inserting a needle into the nucleus pulposus and distending the disc with an injection of saline or contrast media. Postprocedure computed tomography can be used to highlight the features of internal disc disruption, which is the most commonly known cause of discogenic pain. The clinical history usually includes a deep, dull ache in the low back at the midline, with minimal radiation beyond the gluteal area and rarely to the knees and legs, no particular alleviating position, and pain that may worsen with axial loading. There is no accompanying sensory or motor loss. Provocative discography is widely used to identify the symptomatic intervertebral discs. Internal intervertebral disc derangement and positive provocation discography could be treated with surgical fusion or intradiscal electrothermal therapy. Application of heat to the intervertebral disc structure leads to thermal ablation of the nociceptors. Heat leads to contraction and shrinkage of collagen of the intervertebral disc and a decrease of the volume of the nucleus pulposus, resulting in a debulking of the intervertebral disc. Although intradiscal electrothermal therapy is a very promising procedure to treat discogenic pain, studies on its long-term effects, outcome measures, and cost-effectiveness are needed to validate the efficacy of this technique. Copyright © 2000 by W.B. Saunders Company     

Intervertebral disc a source of pain? Low back pain: problems and future directions--case reports

OBJECTIVE: The objective of this article is to provide evidence supporting the idea that intervertebral disc is a source of low back pain. SUMMARY OF BACKGROUND DATA: Diagnostic tests currently available for diagnosis of a painful disc are inadequate. Treatment protocols for low back pain generally ignore the presence of a painful disc. Pathological processes that may be responsible for discogenic pain are incompletely understood. Without diagnosis and treatment, disc disruption evolves to advanced stages of spinal dysfunction. New treatment modalities are becoming available which if applied early may stop disc disruption. CASE REPORTS: We describe here two case reports where discogenic nature of patients' symptoms was suspected based on patients' history, MRI findings and discography. We highlight the inadequacies of spinal imaging and discography in detecting at painful disc. A treatment (Intradiscal electrothermal therapy) was then directed exclusively to the intervertebral discs. We provide arguments that link discal therapy to resolution of patients' symptoms. Resolution of patients' symptoms after the discal treatment raised our suspicion that pain emanated from the intervertebral discs. CONCLUSIONS: Intervertebral disc is a source of low back pain that is often ignored. No diagnostic test currently exists that can reliably confirm presence of a painful disc. Early diagnosis and treatment of a painful disc may reduce enormous pain and suffering from low back pain.     

Activation of satellite cells in the dorsal root ganglia in a disc-punctured rat model

Background  

The neural mechanisms underlying discogenic low back pain caused by disc degeneration remain unclear. Previous studies demonstrated that satellite cells (SC) play an important role in neuropathic pain.     

Sympathetic afferent units from lumbar intervertebral discs

To clarify the pathomechanisms of discogenic low back pain, the sympathetic afferent discharge originating from the L5-L6 disc via the L2 root were investigated neurophysiologically in 31 Lewis rats. Sympathetic afferent units were recorded from the L2 root connected to the lumbar sympathetic trunk by rami communicantes. The L5-L6 discs were mechanically probed, stimulated electrically to evoke action potentials and, finally, treated with chemicals to produce an inflammatory reaction. We could not obtain a response from any units in the L5-L6 discs using mechanical stimulation, but with electrical stimulation we identified 42 units consisting mostly of A-delta fibres. In some experiments a response to mechanical probing of the L5-L6 disc was recognised after producing an inflammatory reaction. This study suggests that mechanical stimulation of the lumbar discs may not always produce pain, whereas inflammatory changes may cause the disc to become sensitive to mechanical stimuli, resulting in nociceptive information being transmitted as discogenic low back pain to the spinal cord through the lumbar sympathetic trunk. This may partly explain the variation in human symptoms of degenerate discs.     

A prospective randomised study on the long-term effect of lumbar fusion on adjacent disc degeneration

The existence and importance of an accelerated adjacent segment disc degeneration (ASD) after lumbar fusion have previously not been demonstrated by RCTs. The objectives of this study were, to determine whether lumbar fusion in the long term accelerates degenerative changes in the adjacent disc and whether this affects the outcome, by using a prospective randomised design. A total of 111 patients, aged 18–55, with isthmic spondylolisthesis were randomised to exercise (EX, n = 34) or posterolateral fusion (PLF, n = 77), with (n = 37) or without pedicle screw instrumentation (n = 40). The minimum 10 years FU rate was 72%, with a mean FU time of 12.6 years (range 10–17 years). Three radiographic methods of ASD quantification were used, i.e. two digital radiographic measurement methods and the semi quantitative UCLA grading scale. One digital measurement method showed a mean disc height reduction by 2% in the EX group and by 15% in the PLF group (p = 0.0016), and the other showed 0.5 mm more disc height reduction in the PLF compared to the Ex group (ns). The UCLA grading scale showed normal discs in 100% of patients in the EX group, compared to 62% in the PLF group (p = 0.026). There were no significant differences between instrumented and non-instrumented patients. In patients with laminectomy we found a significantly higher incidence of ASD compared to non laminectomised patients (22/47 vs. 2/16 respectively, p = 0.015). In the longitudinal analysis, the posterior and anterior disc heights were significantly reduced in the PLF group, whereas in the EX group only the posterior disc height was significantly reduced. Except for global outcome, which was significantly better for patients without ASD, the clinical outcome was not statistically different in patients with and without ASD. In conclusion, the long-term RCT shows that fusion accelerates degenerative changes at the adjacent level compared with natural history. The study suggests that not only fusion, but also laminectomy may be of pathogenetic importance. The clinical importance of ASD seems limited, with only the more severe forms affecting the outcome.     

椎间盘内射频电刺激定位椎间盘源性腰痛责任椎间盘

目的探讨椎间盘内射频电刺激定位椎间盘源性腰痛责任椎间盘的有效性。方法对78例椎间盘源性腰痛患者,在CT引导下通过椎间盘内射频电刺激定位责任椎间盘,根据椎间盘电刺激诱发试验结果分为A组(椎间盘电刺激试验诱发阳性)及B组(椎间盘电刺激试验诱发阴性)。行椎间盘低温等离子纤维环成形术。以疼痛数字评分法(NRS)评价患者疼痛程度,以改良MacNab评分评价术后患者主观满意度,并进行统计学分析。结果 78例中,A组67例,B组11例。2组患者术前NRS评分差异无统计学意义(P0.05),A组术后1周、1个月及6个月NRS评分均较B组减低(P均0.05)。术后1、3、6个月,A组患者MacNab评分优良率均明显高于B组(P均0.05)。术后无严重并发症发生,仅6例出现穿刺点皮下淤血、14例穿刺点局部疼痛,均在术后2周内自行消失。结论椎间盘电刺激诱发试验阳性患者椎间盘低温等离子纤维环成形术后疼痛缓解程度和患者主观满意度更优;椎间盘内射频电刺激有助于准确定位椎间盘源性腰痛责任椎间盘。     

椎间盘源性腰痛的病理形态学观察

目的观察椎间盘源性腰痛的病理形态学特征,探讨其病理机制。方法选取20例椎间盘源性腰痛的椎间盘手术标本,常规HE染色,光学显微镜观察分析。结果镜下见椎间盘髓核面积减少,髓核中活性软骨细胞少,退变细胞多;纤维环增厚,从内层至外层纤维环可见不同程度的破裂,纤维环软骨细胞减少,软骨基质增多。结论椎间盘源性腰痛的主要致病机制可能是椎间盘形态结构的改变作用。     

Resolving discogenic pain

Recent basic science studies on discogenic low back pain have provided new knowledge about this condition. This paper reviews some of these results and presents an overview of the following findings. The rat lumbar intervertebral disk may be innervated non-segmentally through the paravertebral sympathetic nerve and segmentally through the sinuvertebral nerves, and also by dichotomizing sensory fibers. The exposure of the nucleus pulposus (NP) to the outer annulus fibrosus (AF) may induce nerve injury and ingrowth into the disk. Nerve growth factor (NGF)-sensitive neurons are predominant in the rat intervertebral disk, which indicates that hyperalgesic responses can be induced by inflammation. NGF in the NP may promote axonal growth. Lumbar fusion may inhibit nerve ingrowth into the degenerated disk and reduce the percentage of calcitonin gene related peptide (CGRP)-positive neurons.

     

Sensory innervation of the dorsal portion of the lumbar intervertebral disc in rats

STUDY DESIGN: The vertebral levels of dorsal root ganglia innervating the dorsal portion of the L5-L6 intervertebral disc were investigated in rats using a retrograde transport method. The pathways and functions of nerve fibers supplying the dorsal portion of the disc were determined by denervation and immunohistochemistry. OBJECTIVES: The dorsal portion of the lumbar intervertebral disc has been reported to be innervated segmentally, but anesthetic block of the paravertebral sympathetic trunks and the L2 spinal nerve can relieve discogenic low back pain. In the current study, the sensory innervation of the dorsal portion of the L5-L6 intervertebral disc was investigated, because the disc anatomically corresponds to the L4-L5 disc in humans, and the dorsal portion of the human L4-L5 disc is frequently subject to injury that causes low back pain. METHODS: A retrograde transport of Fluoro-Gold (F-G; Fluorochrome, Denver, CO) was used. Subjects included nontreated control (n = 32) and sympathectomized rats in which paravertebral sympathetic trunks were removed from L2 to L3 (n = 9). In a ventral approach, Fluoro-Gold crystals were placed on the dorsal portion of the L5-L6 disc, and labeled neurons in the bilateral dorsal root ganglia from T10 to L6 were counted. RESULTS: Fluoro-Gold crystals did not leak from the dorsal portion of the L5-L6 disc in 14 of the 32 nontreated rats and in 5 of the 9 sympathectomized rats. These rats were used for analysis. Fluro-Gold-labeled neurons were found in dorsal root ganglia from T13 to L6 in the 14 control rats but only from L2 to L6 in the 5 sympathectomized rats. CONCLUSION: The dorsal portion of the L5-L6 disc of rats was shown to be multisegmentally innervated by the T13 to L6 dorsal root ganglia. The sensory fibers from T13, L1, and L2 dorsal root ganglia were shown to innervate the dorsal portion of the L5-L6 disc through the paravertebral sympathetic trunks. In contrast, those from the L3-L6 dorsal root ganglia may innervate the dorsal portion of the L5-L6 disc through the sinuvertebral nerves.     

Advances in the diagnosis of degenerated lumbar discs and their possible clinical application

Purpose

One possible source of chronic low back pain is a degenerated intervertebral disc. In this review, various diagnostic methods for the assessment of the presence of degenerative changes are described. These include clinical MRI, a number of novel MRI techniques and nuclear magnetic resonance spectroscopy.

Methods

Non-systematic literature review.

Results

Clinical MRI is the most commonly employed technique to determine the general “health status” of the intervertebral disc. Novel MRI techniques, such as quantitative MRI, T1ρ MRI, sodium MRI and nuclear magnetic resonance spectroscopy, are more sensitive in quantifying the biochemical changes of disc degeneration, as measured by alteration in collagen structure, as well as water and proteoglycan loss. As potential future diagnostic alternatives, miniature sensors are currently being developed to measure parameters associated with the disc degeneration cascade, such as intradiscal pressure and PG concentration. However, none of the methods listed above show sufficient specificity to identify a degenerated disc as the actual source of the pain. Provocative discography is the only test aimed at a direct diagnosis of discogenic pain, but it has a high false positive rate and there is some evidence of long-term adverse effects. Imaging techniques have also been tested for this purpose, but their validity has not been confirmed and they do appear to be problematic.

Conclusions

A reliable diagnostic tool that could help a clinician to determine if a disc is the source of the pain in patients with chronic LBP is still not available. New MRI techniques are under investigation that could result in a significant improvement over current methods, particularly as they can allow monitoring, not only of morphological but also of biochemical changes.