فهرست مطالب

Basic Medical Sciences - Volume:20 Issue: 7, Jul 2017

Iranian Journal of Basic Medical Sciences
Volume:20 Issue: 7, Jul 2017

  • تاریخ انتشار: 1396/04/25
  • تعداد عناوین: 15
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  • Thomas Low Tat Kuan, Farahnaz Amini, Marjan Seghayat* Pages 729-738
    Multiple sclerosis is a debilitating disease of the central nervous system. It affects people of all ages but is more prevalent among 20-40 year olds. Patients with MS can be presented with potentially any neurological symptom depending on the location of the lesion. A quarter of patients with MS suffer from bilateral lower limb spasticity among other symptoms.
    These devastating effects can be detrimental to the patient's quality of life. Hematopoietic stem cells (HSCs) have been used as a treatment for MS over the past 2 decades but their safety and efficacy has are undetermined. The objective of this study is to evaluate the feasibility and toxicity of autologous HSCs transplantation in MS. A literature search was done from 1997 to 2016 using different keywords. A total of 9 articles, which met the inclusion and exclusion criteria, were included in this review. The type of conditioning regimen and technique of stem cell mobilization are summarized and compared in this study. All studies reported high-dose immunosuppressive therapy with autologous HSCs transplantation being an effective treatment option for severe cases of multiple sclerosis. Fever, sepsis, and immunosuppression side effects were the most observed adverse effects that were reported in the selected studies. HSCs is a feasible treatment for patients with MS; nevertheless the safety is still a concern due to chemo toxicity.
    Keywords: Efficacy, Feasibility, Hematopoietic stem cell, Multiple Sclerosis, Transplantation, Toxicity
  • Yong Chai, Juhua Xiao, Yunyan Du, Zhipeng Luo, Jun Lei, Shouhua Zhang, Kai Huang* Pages 739-744
    Objective(s)
    Non-invasive treatment options for retinoblastoma (RB), the most common malignant eye tumor among children, are lacking. Epithelial growth factor receptor (EGFR) accelerates cell proliferation, survival, and invasion of many tumors including RB. However, RB treatment by targeting EGFR has not yet been researched. In the current study, we investigated the effect of EGFR down-regulation on RB progression using shRNA lentiviral vectors.
    Materials And Methods
    EGFR expression in Weri-Rb-1 cells was down-regulated by EGFR shRNA-bearing lentiviral vectors. Cell death, proliferation, cell cycle as well as invasion after EGFR down-regulation were determined. Further signaling pathway analysis was done by Western blot.
    Results
    Our results revealed that EGFR shRNA could specifically down-regulate EGFR expression and down-regulation of this protein promoted cell death. Further analysis on cell cycle demonstrated that EGFR down-regulation also suppressed cell proliferation by arresting cells at G1 phase. Invasion analysis showed that EGFR down-regulation suppressed cell invasion and was correlated with alteration in the expression of matrix metalloproteinases 2 and 9. Further signaling pathway analysis revealed that EGFR down-regulation mediated RB progression was through PI3K/AKT/mTOR signaling pathway.
    Conclusion
    Our study revealed that EGFR down-regulation, through the PI3K/AKT/mTOR signaling pathway, could inhibit RB progression by promoting cell death while suppressing cell proliferation and invasion. The findings of our study indicated that down-regulation of EGFR using shRNA lentiviral vectors may offer a novel non-invasive treatment for RB.
    Keywords: Epithelial growth factor Lentiviral vector, Mechanistic target of rapamycin, Phosphatidylinositol kinase, Protein kinase B, Retinoblastoma, ShRNA
  • Sara Joushi, Mahmoud Elahdadi Salmani * Pages 745-752
    Objective(s)
    Epilepsy establishment gives rise to biochemical and morphological changes in the hippocampus. Oxidative stress, morphological changes, and mossy fiber sprouting (MFS) in the hippocampus underpin the epilepsy establishment. Eugenol is the main component of the essential oil extracted from cloves with the potential to modulate neuronal excitability. Therefore, we investigated the effect of eugenol on convulsive behavior, oxidative stress, and histological changes of the hippocampus in lithium- pilocarpine model of epilepsy.
    Materials And Methods
    Male Wistar rats weighing 220–250 g were divided into 4 groups; Control, Pilocarpine, Eugenol-Pilocarpine, and Eugenol. Oxidative stress markers were assayed by a biochemical method. Nissl and Timm staining were used to show neuronal survival and MFS, respectively. Behavioral convulsions were evaluated using the modified Racine scale.
    Results
    Eugenol decreased seizure stage and duration as well as mortality. Neuronal numbers were preserved by eugenol treatment in epileptic animals, while eugenol alone reduced the number by itself in all hippocampal sub-regions including DG, CA3, and CA1. Furthermore, eugenol alone increased MDA, GPx and SOD markers, while it increased MDA not only in combined treatment with pilocarpine but also in pilocarpine-treated animals. In contrast to MFS enhancement in naïve animals, eugenol partially reversed the MFS enhancement induced by pilocarpine.
    Conclusion
    Eugenol could prevent behavioral convulsions and show neuroprotective effects through increasing neuronal survival probably by decreasing MFS and increasing the GPx antioxidant marker.
    Keywords: Epilepsy, Eugenol, Hippocampus, Mossy fiber sprouting, Oxidative Stress
  • Zeynab Mohamadi Yarijani, Ali Pourmotabbed, Tayebeh Pourmotabbed, Houshang Najafi Pages 753-759
    Objective(s)
    Crocus sativus (saffron) has been widely used in traditional medicine. It has also been found to possess many beneficial properties in modern medicine. The most important ingredients of saffron are crocin, crocetin, safranal, and picrocrocin. This study evaluated the protective effects of crocin against the inflammation, oxidative stress, and functional disturbances of the kidney induced by renal ischemia/reperfusion (I/R).
    Materials And Methods
    Different doses of crocin (0, 100, 200, and 400 mg/kg) were administered intraperitoneally 30 min before I/R. The rats of the sham group were also injected with normal saline before the sham surgery. For induction of I/R, both renal artery and vein clamped for 30 min, bilaterally. The I/R-induced renal injuries were assessed by measuring leukocyte infiltration, intercellular adhesion molecule-1 (ICAM-1) and tumor necrosis factor-alpha (TNF-α) mRNA expression levels, malondialdehyde (MDA) and ferric reducing/antioxidant power (FRAP) levels in the kidney tissue, and plasma creatinine and urea-nitrogen concentrations.
    Results
    Except for the tissue level of FRAP which decreased, all other measured parameters increased following I/R induction. Pretreatment with all doses of crocin significantly reduced the severity of these disturbances (PP
    Conclusion
    The present study clearly demonstrated the anti-inflammatory, antioxidant, and protective effects of crocin, a main constituent of saffron, against renal damages resulted from I/R in rats.v
    Keywords: Crocin_Inflammation_Ischemia - reperfusion Leukocyte infiltration_TNF-α_ICAM-1
  • Esmat Radmanesh, Mahin Dianat, Mohammad Badavi, Gholamreza Goudarzi, Seyyed Ali Mard Pages 760-768
    Objective(s)
    Particulate matter (PM) exposure can promote cardiac ischemia and myocardial damage. The effects of PM10 on hemodynamic parameters, lipid peroxidation, and infarct size induced by ischemia-reperfusion injury and the protective effects of vanillic acid (VA) in isolated rat heart were investigated.
    Materials And Methods
    Eighty male Wistar rats (250–300 g) were divided into 8 groups (n=10): Control, Sham, VAc, VA, PMa (0.5 mg/kg PM, intratracheal instillation), PMb (2.5 mg/kg PM, intratracheal instillation), PMc (5 mg/kg PM, intratracheal instillation), and PMc VA (5 mg/kg PM, intratracheal instillation; and 10 mg/kg vanillic acid, gavage for 10 days). PM10 was instilled into the trachea in two stages, within 48 hr. After isolating the hearts and transfer to a Langendorff apparatus, hearts were subjected to 30 min ischemia and 60 min reperfusion. Hemodynamic parameters (±dp/dt, LVSP, LVDP, and RPP), production of lipid peroxidation (MDA), and infarct size were assessed.
    Results
    A significant decrease in ±dp/dt, LVSP, LVDP and RPP occurred in PM groups. A significant increase in MDA and myocardial infarct size occurred in PM groups. A significant increase in LVDP, LVSP, ±dp/dt, RPP and decrease in infarct size, MDA, and myocardial dysfunction was observed in groups that received vanillic acid after ischemia–reperfusion.
    Conclusion
    It was demonstrated that PM10 increases MDA, as well as the percentage of cardiac infarct size, and has negative effects on hemodynamic parameters. This study suggests that vanillic acid may serve as an adjunctive treatment in delaying the progression of ischemic heart disease.
    Keywords: Hemodynamic Parameters, Infarct size, Ischemia–reperfusion, Malondialdehyde Particulate matter, Vanillic-acid
  • Soheyla Akhzari, Hossein Rezvan*, Seyed Masoud Zolhavarieh Pages 769-775
    Objective(s)
    It is now supposed that cytokines released during the burn injuries have a great impact on the immunological and pathological responses after the burn. The main objective of this study was to investigate the expression of some pro-inflammatory genes in the wound, spleen and blood neutrophils during the healing process of burn wounds in a murine model.
    Materials And Methods
    The expression of ten pro-inflammatory genes were examined in wounds, spleens and blood neutrophils of mice with burn injuries treated with either silver sulfodiazine or phosphate-buffered saline (PBS) using RT-PCR at the end of the first and second weeks after injuries.
    Results
    None of the pro-inflammatory genes were expressed in the skin, spleen and blood neutrophils of healthy mice. In the group control, IL-12P35, IL-12P40, CCR5, IL-1β and IFN- γ were expressed in the spleen and blood neutrophils in the first week. Instead, CCL5, CCR5, IL-1β and IFN- γ were expressed in the wound, but in the second week, the expression of the genes became similar. In the test group, in the first week, TNF-α, IL-12P35, IL-12P40 and IL-1β were expressed in the lesions, CCL4, IL-1α, IL-12P35, IL-12P40, CCR5 and IFN- γ were expressed in the spleen and no pro-inflammatory gene expression was detected in blood neutrophils.
    Conclusion
    IL-1β and IFN- γ are expressed in wound, spleen and neutrophils of untreated mice, but not in silver sulfodiazine treated mice. Hence, treatment with silver sulfodiazine suppressed the expression of pro-inflammatory genes in some stages of healing.
    Keywords: Burn, Neutrophils, Pro-inflammatory, Silver sulfodiazine, Spleen, wound
  • Mohammad Naser Shafei, Tahereh Nikyar, Mahmoud Hosseini, Saeed Niazmand, Maryam Paseban Pages 776-782
    Objective(s)
    Nitric oxide (NO) is an important neurotransmitter in central nervous system involved in central cardiovascular regulation. The presence of NO in the pedunculopontine tegmental (PPT) nucleus has been shown, but its cardiovascular effect has not been determined. In the present study, the cardiovascular effect of NO in the PPT nucleus was evaluated.
    Materials And Methods
    After induction of anesthesia, a polyethylene catheter (PE-50) filled with heparinized saline inserted into the femoral artery, and the blood pressure (BP) and heart rate (HR) were continuously recorded. Animals were then placed in a stereotaxic apparatus and maximum changes of mean arterial pressure (∆MAP) and heart rate (∆HR) after microinjection of two doses of NG-nitro-L-arginine methyl ester (L-NAME, 30 and 90 nmol), L-arginine (L-Arg 10 and 50 nmol) and sodium nitroprusside (SNP, 9 and 27 nmol) into the PPT were provided and compared with control group (One-way ANOVA).
    Results
    Both doses of L-NAME significantly increased ∆MAP compared to control (PP
    Conclusion
    Our results show an inhibitory effect of the nitrergic system of the PPT on central cardiovascular system.
    Keywords: blood pressure, L-NAME, Microinjection, Nitric oxide, Pedunculopontine tegmental, Sodium nitroprusside
  • Liang Wang, Jinhong Wu, Changao Xie* Pages 783-790
    Objective(s)
    MicroRNAs (miRNAs) are considered as powerful, post-transcriptional regulators of gene expression in hepatocellular carcinoma cells (HCC). However, the function of miR-92a is still unclear in HCC.
    Materials And Methods
    Expression of miR-92a in human HCC cell lines was evaluated using qRT-PCR. MTT assay and transwell assay were used to examine the function of miR-92a in HepG2 and Huh7 cells. Bioinformatic analyses and luciferase reporter assays were used to validate FOXA2 as a direct target gene of miR-92a. Consistently, the biological outcome of miR-92a on regulating FOXA2 was examined by proliferation and invasion analysis in vitro.
    Results
    Here, we detected the higher expression of miR-92a in human HCC cell lines, such as HepG2, Huh7 and Hep3B, compared with the normal human hepatocyte L02 cells. Overexpression of miR-92a significantly increased cell growth and invasion ability, while the knockdown of miR-92a could remarkably inhibit the growth and invasion possibility. We identified that miR-92a has specific targeting sites in the 3'-UTR of the FOXA2. By overexpressing miR-92a in HepG2 cells or Huh7 cells, the expression of FOXA2 was remarkably repressed.
    Conclusion
    We demonstrated that miR-92a may play a critical role in HCC proliferation and invasion and may serve as a novel therapeutic target by the repression of FOXA2.
    Keywords: miR-92a promotes HCC proliferation, invasion
  • Mohsen Khaki, Ali Hatef Salmanian, Ghasem Mosayebi, Maryam Baazm, Saeed Babaei, Neda Molaee, Hamid Abtahi * Pages 791-797
    Objective(s)
    Vascular endothelial growth factor (VEGF) is one of the most effective proteins in angiogenesis, mesenchymal stem cells (MSCs) differentiation and wound healing. These abilities are therapeutic potential of VEGF in diabetic retinopathy, nephropathy and other tissue damage circumstances. In this study, recombinant VEGF was produced in Escherichia coli (E. coli) system and then biological activity of this protein was evaluated in animal wound healing.
    Materials And Methods
    E. coli BL21 (DE3) competent cells were transformed with pET32a-VEGF clone and induced by isopropyl-β-D-thio-galactoside (IPTG). The recombinant protein was purified byaffinity chromatography. Recombinant VEGF-A-based ointment (VEGF/Vaseline 0.8 mg/100 w/w) was used for external wound (25×15mm thickness) healing in animal model. In vivo activity of ointment was evaluated by clinical evidences and cytological microscopic assessment.
    Results
    The recombinant protein with molecular weight of 45 kilodaltons (kDa) and concentration of 0.8 mg/ml was produced.Immunoblotting data showed that the antigenic region of VEGF can be expressed in E. coli and the recombinant protein has similar epitopes with close antigenic properties to the natural form. Macroscopic findings and microscopic data showed that the recombinant VEGF-A ointment was effective on excisional wound healing.
    Conclusion
    Recombinant VEGF-A produced by pET32a in E. coli, possesses acceptable structure and has wound healing capability.
    Keywords: Biological activity, Recombinant, VEGF-A, Wound healing
  • Roodabeh Bahramsoltani, Mohammad Hosein Farzaei, Amir Hossein Abdolghaffari, Roja Rahimi, Nasrin Samadi, Mohammad Heidari, Mohammadamin Esfandyari, Maryam Baeeri, Gholamreza Hassanzadeh, Mohammad Abdollahi, Saba Soltani, Ali Pourvaziri, Gholamreza Amin * Pages 798-805
    Objective(s)
    Cucurbita moschata Duchesne (pumpkin) is a well-known plant with several pharmacological effects. The aim of the present study was to assess burn wound healing activity of C. moschata peel extract (CE). Also, standardized CE was assessed for antioxidant activity and antibacterial effects against major pathogens of burns.
    Materials And Methods
    Healing properties of topical preparation of 10% and 20% concentrations of CE were assessed on second degree burn in rats during a 14-day period as well as histological studies, total antioxidant power, lipid peroxidation and total thiol content of skin tissue samples.
    Results
    Radical scavenging IC50 and ferric-reducing antioxidant power value were 4.015±0.20 mg/ml and 142.63±2.65 mmol Fe2, respectively. Total mucilage content was 13.8%. The optimal results were obtained by 20% CE that showed 90.80±5.86 % wound closure and tissue repair as well as significant reduction of tissue oxidative stress biomarkers. Histological analyses confirmed wound healing activity of pumpkin peel extract.
    Conclusion
    Considering the high mucilage content of the plant, providing a moist environment for wound, C. moschata peel extract could be a natural remedy for treatment of burns. Further clinical studies are suggested to confirm C. moschata peel extract as a wound healing agent.
    Keywords: Cucurbita moschata, Histology, Traditional Persian Medicine, Pumpkin, Wound healing
  • Mahboobe Akbari, Mohammad Khaksari, Maryam Rezaeezadeh-Roukerd, Moghaddameh Mirzaee, Mahdieh Nazari-Robati * Pages 806-812
    Objective(s)
    Chondroitinase ABC (cABC) treatment improves functional recovery following spinal cord injury (SCI) through degrading inhibitory molecules to axon growth. However, cABC involvement in other pathological processes contributing to SCI remains to be investigated. Here, we studied the effect of cABC I on oxidative stress and inflammation developed in a rat model of SCI.
    Materials And Methods
    Male rats (220–250 g) were divided into three groups (n=28) including rats that underwent SCI (SCI group), rats subjected to SCI and received an intrathecal injection of phosphate buffer saline (SCI㰔 group), and rats that underwent SCI and received cABC intrathecally (SCI group). Then, the level of TNF-α, Il-1β, malondialdehyde, nitric oxide, and myeloperoxidase in injured tissues, as well as hindlimb motor function, were measured at 4 hr, 1, 3 and 7 days post-SCI.
    Results
    Our data showed that cABC treatment reduced the development of inflammation and oxidative stress associated with SCI at all-time points. In addition, functional recovery was improved in rats that received cABC at 7 days post-SCI.
    Conclusion
    The present findings indicate that cABC treatment can exert its neuroprotective effect through modulation of post-traumatic inflammatory and oxidative response.
    Keywords: Chondroitinase ABC, Functional recovery, Inflammation, Oxidative Stress, Spinal cord injury
  • Lihua Liu, Zhongfu Zuo, Sijing Lu, Aihua Liu, Xuezheng Liu * Pages 813-821
    Objective(s)
    Naringin, an essential flavonoid, inhibits inflammatory response and oxidative stress in diabetes. However, whether naringin has beneficial effects on diabetic retinopathy (DR) remains unknown.
    Materials And Methods
    Streptozotocin (STZ, 65 mg/kg) was intraperitoneally injected into male rats (8 weeks old weighting 200-250 g) to establish diabetic model, then naringin (20, 40 or 80 mg/kg/day) was intraperitoneally injected into the diabetic rats for twelve weeks. Glial fibrillary acidic protein (GFAP) level, thickness of ganglion cell layer (GCL) and ganglion cell counts were assessed in diabetic retina in vivo. Naringin (50 μM) that significantly inhibited high glucose (HG, 25 mM)-induced cell proliferation was used to treat rat Muller cell line (rMC1) in vitro. Inflammatory response, oxidative stress and activation of nuclear factor kappa B (NF-κB) p65 were evaluated in retina in vivo and in rMC1 cells in vitro.
    Results
    Naringin alleviated DR symptoms as evidenced by the increased retinal ganglion cells and decreased GFAP level in rat retina. Naringin exhibited anti-inflammatory and antioxidative effects as confirmed by the down-regulated pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), and the up-regulated antioxidants, glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) in DR rats. Moreover, we found that naringin inhibited HG-induced proliferation, abnormal inflammatory response and oxidative stress in rMC1 cells. In addition, the enhanced nuclear translocation of NF-κB p65 in diabetic rat retina and HG-induced rMC1 cells was suppressed by naringin.
    Conclusion
    Naringin attenuates inflammatory response, oxidative stress and NF-κB activation in experimental models of DR.
    Keywords: Diabetic Retinopathy, Inflammation, Naringin, NF-?B pathway, Oxidative Stress
  • Firoozeh Alavian *, Saeedeh Ghiasvand Pages 822-828
    Objective(s)
    Central nucleus of amygdala (CeA) is the most important region for morphine-induced reward, and GABAergic system plays an important role on morphine reinforcement. The influence of CeA administration of GABAB receptor agonist and antagonist on the expression and acquisition of morphine-induced incentive tolerance using conditioned place preference (CPP) paradigm was investigated in the present study. Our purpose was to evaluate the role of CeA GABAB receptors in morphine tolerance.
    Materials And Methods
    Seven days after surgery and cannulation, the experiments were begun. Subcutaneous (SC) injections of morphine induced CPP. Administration of one daily dose of morphine (12.5 mg/kg) for 3 days in order to develop tolerance to the drug reduced the conditioning induced by morphine (7.5 mg/kg, SC). GABAB receptor agonist, baclofen (1.5, 6 and 12 µg/rat) or GABAB receptor antagonist, CGP35348 (1.5, 6 and 12 µg/rat) were injected into the CeA 5 min before the experiments in the test day (expression of tolerance) or 5 min before each injection of morphine (12.5 mg/kg) (acquisition of tolerance).
    Results
    It was shown that injections of baclofen (1.5 and 12 µg/rat) reduced acquisition, whereas the dose of 6 µg/rat of the drug exacerbated the acquisition of morphine tolerance. Baclofen at all doses significantly increased the expression of tolerance to morphine. Administration of CGP35348 (1.5, 6 and 12 µg/rat) reduced the acquisition and expression of morphine tolerance.
    Conclusion
    These results confirmed the importance of GABAB receptors with in the CeA in morphine tolerance in female rats.
    Keywords: Central nucleus of amygdala, Conditioned place preference, GABAB receptors, Morphine, Tolerance
  • Narges Khaghanzadeh, Afshin Samiei, Zahra Mojtahedi, Mohammad Ramezani, Massood Hosseinzadeh, Abbas Ghaderi * Pages 829-834
    Objective(s)
    Umbelliprenin is a prenyloxy-coumarin with pharmacologically polyvalent activity. Several studies have shown its anti-inflammatory, anti-tumor, antioxidant, and antigenotoxic activity, and other functions. However, the exact mechanism of action of this compound on the immune response has not yet been shown. Here, we investigated umbelliprenin effects on the predominance of Th1 and Th2 responses in normal C57/BL6 mice.
    Materials And Methods
    Umbelliprenin (2.5 mg/200 µl IP) were administered to six C57/BL6 mice every other day for 8 days. Paraffin and PBS-injected mice were enrolled as solvent and control groups, respectively (n=6 mice/group). IL-10, IFN-γ, and IL-4 levels were determined in sera and also in splenocytes culture supernatants in the presence of Con A (3 µg/ml) after 72 hr. H&E staining of paraffin embedded blocks was performed for lung and liver tissues of mice.
    Results
    Umbelliprenin could significantly increase the secretion of IFN-γ and IL-4 in sera and IL-10 in splenocytes cultures. Comparison of IFN-γ /IL-4 in the sera and splenocytes culture supernatants showed lower ratios in umbelliprenin treated mice than in solvent and untreated groups.
    Conclusion
    The in vivo study showed that umbelliprenin could induce anti-inflammatory responses via the predominance of Th2 cells and some regulatory responses in C57/BL6 mice.
    Keywords: Anti-inflammatory_Concanavalin A (Con A)_Interferon gamma (IFN-?)_Prenyloxy-coumarin_Umbelliprenin
  • Farideh Vahabi, Sedigheh Sadeghi, Mohammad Arjmand, Fatemeh Mirkhani, Eshagh Hosseini, Mahsheed Mehrabanfar, Reza Hajhosseini, Ayda Iravani, Parastou Bayat, Zahra Zamani * Pages 835-840
    Objective(s)
    Determination of stages of colon cancer is done by biopsy usually after surgery. Metabolomics is the study of all the metabolites using LC-MS and 1HNMR spectroscopy with chemometric techniques. The stages of colon cancer were detected from patient's sera using 1HNMR.
    Materials And Methods
    Five ml blood was collected from 16 confirmed patients referred for colonoscopy. One group of eight patients were diagnosed with stage 0 to I colon cancer and the second group of 8 patients with II-IV stage colon cancer. Sera were sent for 1HNMR. The differentiating metabolites were identified using HMDB and the metabolic cycles from Metaboanalyst.
    Results
    Six metabolites of which pyridoxine levels lowered, and glycine, cholesterol, taurocholic acid, cholesteryl ester and deoxyinosine increased.
    Conclusion
    The different stages of cancer were identified by the main metabolic cycles such as primary bile acid biosynthesis, purine and vitamin B metabolic pathways and the glutathione cycle.
    Keywords: Colon cancer, HNuclear Magnetic Resonance Spectroscopy, Metabolomics, Serum, Staging