|Year : 2020 | Volume
| Issue : 2 | Page : 90-95
Histomorphological characterization of Pneumococcal meningitis in bone marrow of Wistar rats treated with Bridelia ferruginea and ciprofloxacin
Olusegun Dare Omotoso1, Abdullahi A Alfa2, Samson E Olorunnado1, Kokori B Tijani3
1 Department of Anatomy, Faculty of Basic Medical Sciences, Kogi State University, Anyigba, Nigeria
2 Department of Microbiology, Faculty of Sciences, Kaduna State University, Kaduna, Nigeria
3 Department of Pharmacology and Therapeutics, Faculty of Basic Clinical Sciences, Kogi State University, Anyigba, Nigeria
|Date of Submission||11-Jan-2020|
|Date of Decision||08-Apr-2020|
|Date of Acceptance||14-Jun-2020|
|Date of Web Publication||29-Jul-2020|
Olusegun Dare Omotoso
Department of Anatomy, Faculty of Basic Medical Sciences, Kogi State University, Anyigba.
Source of Support: None, Conflict of Interest: None
Background: Bridelia ferruginea is one of the most valuable ethnomedicinal plants, which has been confirmed for antimicrobial activities and tissue-enhancing properties against degenerative processes. This study aimed at investigating the histomorphological characterization of ethanol extracts of leaf, stem-bark, and root of B. ferruginea on Pneumococcal-meningitis-induced bone marrow damage. Materials and Methods: Sixty-three Wistar rats were randomly divided into nine groups of seven rats each. Group B-F2 were inoculated with 1 × 109 colony-forming units (CFU)/mL of P. meningitis and were left for 72h before treatment. Group A rats (normal control) received 2.5 mg/kg of phosphate-buffered saline (PBS), whereas group B rats (negative control) were left untreated, group C rats (positive control) were treated with single dose daily of 1.4 mg/kg ciprofloxacin tablets, group D1 received 300 mg/kg B. ferruginea leaf extract, group D2 received 600 mg/kg B. ferruginea leaf extract, group E1 received 300 mg/kg B. ferruginea stem-bark extract, group E2 received 600 mg/kg B. ferruginea stem-bark extract, group F1 received 300 mg/kg B. ferruginea root extract, and group F2 received 600 mg/kg B. ferruginea root extract for 2 weeks after which all the animals were killed via cervical dislocation. Results: Ciprofloxacin and the leaf, stem-bark, and root extracts of B. ferruginea were able to avert and managed damage (tissue damage, histomorphological, and cytoarchitectural parameter) caused by P. meningitis in the bone marrow of adult Wistar rats. Conclusion: Bridelia ferruginea has antibacterial activities against factors causing bone marrow erythyroid hyperplasia, osteomyelitis as well as hemolytic anemia.
Keywords: Bone marrow, Bridelia ferruginea, erythyroid hyperplasia, Pneumococcal meningitis
|How to cite this article:|
Omotoso OD, Alfa AA, Olorunnado SE, Tijani KB. Histomorphological characterization of Pneumococcal meningitis in bone marrow of Wistar rats treated with Bridelia ferruginea and ciprofloxacin. Int J Med Health Dev 2020;25:90-5
|How to cite this URL:|
Omotoso OD, Alfa AA, Olorunnado SE, Tijani KB. Histomorphological characterization of Pneumococcal meningitis in bone marrow of Wistar rats treated with Bridelia ferruginea and ciprofloxacin. Int J Med Health Dev [serial online] 2020 [cited 2020 Oct 25];25:90-5. Available from: https://www.ijmhdev.com/text.asp?2020/25/2/90/291060
| Introduction|| |
Diseases of the blood, bone marrow, and brain are among the most leading and debilitating infections of the recent century. The most severe acute bacterial disease of the bone marrow and blood is osteomyelitis, erythyriod hyperplasia, and hemolytic anemia, respectively, caused by Streptococcus pneumoniae––pneumococcal meningitis––a disease world spread among children and adults. The death rate from this disease is between 15% in industrialized and 40% in developing countries., Autopsy examination of patients who died following P. meningitis revealed cerebral edema, cerebral infarctions and hemorrhages, osteomyelitis, apoptosis and necrosis of the hippocampal dentate gyrus and bone marrow,,. Some of these pathological attributes have been reproduced in rat models, which provide the condition for novel drug development and pathophysiological investigation., Several murine models have been developed, using intracerebral,, intraperitoneal, intravenous, intranasal, or intracisternal methods of inoculation. There is a continuing need for the development of new treatment strategies such as drug, vaccine from plant of ethno medicinal importance.
Ethnomedicinal plants are used worldwide and have an increasing economic importance. The use of plant parts to manage human disease is as old as the disease itself and herbal plant was the major form of medicine in Africa especially in Nigeria. In addition, 80%–90% of developing countries population depends on traditional medicines for their primary health care and 30%–40% of the drugs are based on plants and their derivatives.Bridelia ferruginea appears to be the most studied species regarding both their ethno medicinal and ethno pharmacological properties., According to ethnomedicinal properties surveys of B. ferruginea in African countries, these plant species are used as antimicrobial against osteomyelitis and meningitis in traditional human medicine.,,Bridelia ferruginea Benth (Family: Euphorbiaceae) (BF) is a widely used ethnomedicinal plant in several parts of Africa for the treatment of various ailments. It is usually used in the form of decoctions and extracts of parts of the plant and is about the most studied of the species Bridelia. In Nigeria, it is commonly found in the Savannah and other more humid regions of Africa. The aqueous stem-bark extract has been reported to show anti-inflammatory, antipyretic, and analgesic activities in vivo and in vitro as well as significant antiplasmodial activity against plasmodium berghei berghei in mice. This study investigated the curative and management measure against osteomyelitis and erythyriod hyperplasia in bone marrow of rat models inoculated with P. meningitis using B. ferruginea triplicate extracts as against standard antibacterial drug (ciprofloxacin).
| Materials and Methods|| |
Bridelia ferruginea leaves, roots, and stem-bark were harvested from Kogi State University Farm in Anyigba, Nigeria and the plant regimens were identified with voucher number (No. FHI. 110342) assigned at Forestry Research Institute of Nigeria (FRIN), Jericho hill, Ibadan, where it was deposited.
Plants extraction and preparation
The leaves, roots, and stem bark of fresh B. ferruginea plant were plucked and air dried under room temperature at 29–35°C for 8 weeks, after which they were pulverized into coarse form each with Acrestor high-speed milling machine. The coarse forms (200 g) were macerated in absolute ethanol. They were left to stand for 24h. After that the extracts were filtered through muslin cloth on a plug of glass wool in a glass column. The resulting ethanol extracts were concentrated and evaporated to dryness using rotary evaporator at an optimum temperature which was between 40 and 45°C to avoid denaturation of the active ingredients. The concentrated extract was store in the refrigerator until use.
Source and methods of Pneumococcal meningitis inoculation
A clinical isolate of S. pneumoniae serotype 3 was obtained from Amahdu Bello University Teaching Hospital, Zaria, Nigeria, and was grown to mid log phase in 4h at 37°C in Todd Hewitt broth supplemented with 0.5% yeast extract. At an OD620 of 0.8–1.0 the S. pneumoniae were centrifuged and washed twice by resuspension in sterile 0.9% NaCl and recentrifugation. Finally, the bacteria were resuspended in sterile NaCl 0.9% to yield an approximate concentration of 1 × 109 colony-forming units (CFU)/mL. The exact number of CFUs was subsequently determined for inoculates by serial dilution method and on blood agar plates (overnight at 37°C). The experimental rats were inoculated with 1 × 109 CFU/mL (0.1mL) through intranasal using nasopharyngeal tube; the animals were left for 72h before the commencement of treatment.
Animal ethical clearance
All experimental investigations were done in compliance with the guideline, as stated in the “Guide to the care and use of Laboratory Animals Resources” and in accordance with guidelines stated in IACUC and OLAW, United Kingdom.
Conditioning of animals
Animals were bred in the animal house of the College of Health Sciences, Kogi State University, Anyigba, Nigeria, to rule out the genetic effects on the investigation and second filial generation were used for this study. The study was carried out using healthy adult Wistar rats of both sexes weighing 120–160 g. The animals were maintained under standard laboratory conditions of light, temperature, humidity, and ventilation. They were given rat chow and water ad libitum and the experimental animals were acclimatized for 2 weeks before the commencement of the research work.
A total of 63 adult Wistar rats aged 12 weeks of both sexes were used for this study. The animals were randomly divided into nine groups A, B, C, D1, D2, E1, E2, F1, and F2 of seven Wistar rats each [Table 1].
This treatment was through gavage, a single dose per day for the period of 2 weeks.
Animal killing and histological analysis
Twenty-four hours after the last administration, all animals were killed via cervical dislocation. The long bones from all the rats were carefully excised from the limbs using forceps, weighed and both the proximal and the distal ends of the bones were carefully cut open and normal saline was used to aspirate the bone marrow from all the long bones of the animals were preserved in 10% formol-saline. After 24h of fixation, the bone marrow of all long bones tissues were routinely processed for hematoxylin and eosin (H & E).
The bone marrow tissue photomicrographs were captured using trioccular microscope coupled with scope image 9.0 (HIC) camera with specification (HDCE-10C (G012012536), made in Germany.
| Results and Discussions|| |
Hematoxylin and eosin (H & E) bone marrow histology in rats inoculated with P. meningitis treated with B. ferruginea plant extracts.
| Discussion|| |
Bone marrow is one of the largest organs in the body. It is the major hematopoietic organ and is important as both a primary and secondary lymphoid organ. As the hematopoietic system is a potential target organ of chemical and microbial exposure, evaluation of the blood and bone marrow is an important component of toxicity or safety assessment studies. As part of the hematopoietic system evaluation, morphological assessment of bone marrow tissue sections provides information about bone marrow tissue architecture (e.g., cellularity, cell lineages, vascular or stromal alterations, inflammation and necrosis), estimation of iron stores and identification of other features (e.g., pigment, infectious agents, proliferative or neoplastic disorders). The use of ethno-therapeutic plants in the primary treatment of ailments among Africa countries is of high economic importance and has provided easy accessibility and cost-effective to the populace. Bridelia ferruginea as one of the medicinal plants of interest has been reported in various studies of its activities in treatment and management of various diseases. Its antimicrobial, anti-inflammatory, and antiplasmodial properties have been reported in several investigations. This study revealed the antibacterial activities of B. ferruginea leaf, stem bark, and root in treatment and management of bone marrow: P. meningitis-related diseases and osteomyelitis as compared with standard drug (ciprofloxacin) use in treating bacterial and microbial infections. Increased cell number in nucleated cells in histological sections of bone marrow usually indicates a response to increased cell demand and is most evident in the hematopoietic cell lineage of the negative control rat models as definitive identification of lymphoid cells is not possible in H&E-stained preparations. Hematopoietic cell increases (also has been referred to as hypercellularity, hematopoietic cell hyperplasia or hematopoietic cell proliferation) are diagnosed by estimating the percentage of the marrow cavity containing fat versus hematopoietic cells and comparing these percentages to concurrent control animals [Plate A]. In rodents, if the hyperplasia is marked, hematopoietic cells may fill the marrow space, even extending through the nutrient foramina [Plate B]. In adult dogs, marrow hyperplasia should be considered when greater than 75% of the marrow space is occupied by hematopoietic cells. Hematopoietic cell increases may involve all cell lines (panhyperplasia) or individual cell lines. When only one line is involved, the hematopoietic cell increases could indicate the specific lineage affected (e.g., erythroidormyeloid). Cell morphology is usually unaltered and the maturation sequence is synchronous; though, scattered atypical cells (e.g., binucleate rubricytes, giant band neutrophils, Howell–Jolly bodies) may be observed related to the increased production demand., Depending on the cell line affected there may also be evidence of a shift in the myeloid/erythroid (M:E) ratio. For example, an increase in erythroid cells would decrease the estimated M:E ratio. Other cellular elements of the bone marrow such as mast cells and megakaryocytes can also be increased in number. Increases in mast cell numbers (mastocytosis) have, however, also been observed in the bone marrow sections of [Plate B] rats. Emperipolesis of neutrophils within megakaryocytes has been observed in rats and mice and has been associated with administration of a growth factor or hematopoietic cell hyperplasia related to chronic blood loss or inflammation., Although histologic sections may provide evidence of the linage affected, smears or cytocentrifuge preparations of bone marrow are usually necessary for accurate determination of the M:E ratio and synchrony of maturation. In most species, increases in erythropoietic cells (erythroid hyperplasia) [Plate B] usually suggest a response to anemia (e.g., early blood loss or hemolytic anemia). Histologic sections plate treated with B. ferruginea leaf, stem bark, and root at various doses shown in plates C, D1, D2, E1, E2, F1 and F2 established the efficacy of the plant in the treatment of bone marrow microbial infections as compared with the histologic sections of normal control and positive control as against the negative control rats. [Plate C], [Plate D1], [Plate D2], [Plate E1], [Plate E2], [Plate F1] and [Plate F2] shows marrow populated by numerous monotonous appearing hemopoietic precursor cells admixed with interspersed larger megakaryocytes which is an indication ameliorative measure on histomorphological damage caused by P. meningitis in the bone marrow of Wistar rats.
|Plate A: (MAG X 400): Histologic section of tissue showing marrow populated by numerous monotonous appearing hemopoietic precursor cells admixed with interspersed larger megakaryocytes. Normal bone marrow histology|
Click here to view
|Plate B: (MAG X 400): Histologic section of tissue showing marrow with less monotonous appearing hemopoietic precursor cells admixed with cluster of large megakaryocyte, indicative of erythyriod hyperplasia. Abnormal bone marrow histology|
Click here to view
|Plate C: (MAG X 400): Histologic section of tissue showing marrow populated by moderate monotonous appearing hemopoietic precursor cells admixed with interspersed larger megakaryocytes. Relatively normal bone marrow histology|
Click here to view
|Plate D1: (MAG X 400): Histologic section of tissue showing marrow populated by numerous monotonous appearing hemopoietic precursor cells admixed with interspersed larger megakaryocytes. Normal bone marrow histology|
Click here to view
|Plate D2: (MAG X 400): Histologic section of tissue showing marrow populated by numerous monotonous appearing hemopoietic precursor cells admixed with interspersed larger megakaryocytes. Normal bone marrow histology|
Click here to view
|Plate E1: (MAG X 400): Histologic section of tissue showing marrow populated by moderate monotonous appearing hemopoietic precursor cells admixed with interspersed larger megakaryocytes. Normal bone marrow histology|
Click here to view
|Plate E2: (MAG X 400): Histologic section of tissue showing marrow populated by numerous monotonous appearing hemopoietic precursor cells admixed with interspersed larger megakaryocytes. Normal bone marrow histology|
Click here to view
|Plate F1: (MAG X 400): Histologic section of tissue showing marrow populated by moderate monotonous appearing hemopoietic precursor cells admixed with interspersed larger megakaryocytes. Normal bone marrow histology|
Click here to view
|Plate F2: (MAG X 400): Histologic section of tissue showing marrow populated by numerous monotonous appearing hemopoietic precursor cells admixed with interspersed larger megakaryocytes. Normal bone marrow histology|
Click here to view
| Conclusion|| |
This study has revealed the following conclusions:
- Bridelia ferruginea leaf, stem bark, and root extracts have high level of bioactive constituents as well as natural antioxidants, which has been reported in our previous published articles and is useful to the body system in maintenance of body defense mechanism against stress and factors causing diseases.
- It has been found that B. ferruginea leaf, stem bark, and root have ameliorative measure on histomorphological damage caused by P. meningitis in the bone marrow of Wistar rats.
- This study has given a better understanding that P. meningitis can cause erythyroid hyperplasia, which may result into hemolytic anemia and osteomyelitis in bone marrow as evident in the reported photomicrograph.
- Bridelia ferruginea plant should be subjected into more studies involving clinical trials.
- Bridelia ferruginea plant efficacy and medicinal importance should be advocated and more public awareness should be encouraged on the use of the plant extract.
- Development of more potent drugs and vaccine against P. meningitis should be encouraged.
- Raise public awareness regarding sources of exposure to P. meningitis stating the risk mitigation measure.
- Adequate and good monitoring and workable health system should be made available and accessible to workers or people living in areas contaminated with microbial activities.
- Research in phytochemical therapy for neurological diseases should be encouraged and government should made more financial aids available for researchers in this area of study.
Financial support and sponsorship
| References|| |
Lagi F, Bartalesi F, Pecile P, Biagioli T, Caldini AL, Fanelli A, et al
. Proposal for a new score-based approach to improve efficiency of diagnostic laboratory workflow for acute bacterial meningitis in adults. J Clin Microbiol 2016;54:1851-4.
Resti M, Moriondo M, Cortimiglia M, Indolfi G, Canessa C, Becciolini L, et al
. Community-acquired bacteremic Pneumococcal pneumonia
in children: Diagnosis and serotyping by real-time polymerase chain reaction using blood samples. Clin Infect Dis 2010;51:1042-9.
Mbah CC, Akuodor GC, Anyalewechi NA, Iwuanyanwu TC, Osunkwo UA In vivo antiplasmodial activities of aqueous extract of Bridelia ferruginea
stem bark against plasmodium berghei berghei in mice. Pharm Biol 2012;50:188-94.
Mook-Kanamori BB, Geldhoff M, van der Poll T, van de Beek D Pathogenesis and pathophysiology of Pneumococcal meningitis
. Clin Microbiol Rev 2011;24:557-91.
Kokori BT, Abdullahi AA, Abdullahi AS Studies on phytochemical, nutraceutical profiles and potential medicinal values of Allium sativum Linn (Lilliaceae) on bacterial meningitis. Int Neuropsychiatr Dis J 2019;13:1-15.
Borsa N, Di Pasquale M, Restrepo MI Animal models of Streptococcus pneumoniae
disease. Int J Mol Sci 2019;20:1-20.
Foucar K, Reichard K, Czuchlewski D Bone marrow pathology. 3rd ed. Vols 1 and 2. Chicago, IL: ASCP Press; 2010.
Hackam DG, Wooward M, Newby LK, Bhatt DL, Shao M, Snuth EE, et al
. European publication, pathogenesis and pathophysiology of hemorrhage, bacterial endotoxin in vital organs and cerebral hemorrhagic stroke. Eur J NeuroSci 2011;124:2233-42.
Wang Y, Jiang B, Guo Y, Li W, Tian Y, Sonnenberg GF, et al
. Cross-protective mucosal immunity mediated by memory th17 cells against Streptococcus pneumoniae
lung infection. Mucosal Immunol 2017;10:250-9.
Janesch P, Stulik L, Rouha H, Varga C, Steinhäuser J, Zerbs M, et al
. Age-related changes in the levels and kinetics of pulmonary cytokine and chemokine responses to Streptococcus pneumoniae
in mouse pneumonia models. Cytokine 2018;111:389-97.
Majhi A, Kundu K, Adhikary R, Banerjee M, Mahanti S, Basu A, et al
. Combination therapy with ampicillin azithromycin in an experimental Pneumococcal pneumonia
is bactericidal effective in down regulating inflammation in mice. J Inflamm (Lond.) 2014;11:5.
Akudor GC, Mbah CC, Anyalewechi NA, Idris-Usman M, Iwuanyanwu TC, Osunkwo UA Pharmacological profile of aqueous extract of Bridelia ferruginea
stem bark in the relief of pain and fever. J Med Plants Res 2011;5:5366-9.
Aziz MA, Bashar K, Chowdhury MMH, Faruque A Antibacterial, toxicity and larvicidal potentiality of microcos paniculata barks. Pharmacol Online Arch 2015;1:109-20.
Gong F, Gu H, Xu Q, Kang W Genus mitragyna: Ethnomedicinal uses and pharmacological studies. Phytopharmacology 2012;3:263-72.
Alfa AA, Abimbola Ayodeji O, Donatus Teru GA, Bajeh Tijani K Studies on the phytochemical compounds in the Ethanolic Leaf Extract (ELE), Ethanolic Bark Extract (EBE) and Ethanolic Root Extract (ERE) of Bridelia ferruginea
benth (Euphorbiaceae). Asian J Biochem Genet Mol Biol 2019;2:1-8.
Djoueché CM, Azebaze AB, Dongmo AB Investigation of plants used for the ethnoveterinary control of gastrointestinal parasites in Bénoué Region, Cameroon. Tropicultura 2011;29:205-11.
Ajibesin KK, Edulis D, Lam HJ A review on its medicinal, phytochemical and economical properties. Res J Med Plant 2011;5:32-41.
Yadav V, Sharma L, Thomas B, Al Hail M. An overview on nutraceuticals as pharmacological agents. Adv Biores 2012;3:113-28.
Shakya AK Medicinal plants: Future source of new drugs. Int J Herbal Med 2016;4:5964.
Olumayokun AO, Mutalib AA, Uchechukwu PO, Bernd LF Bridelia ferruginea produces antineuroinflammatory activity through inhibition of nuclear factor- KappaB and P38 MAPK signaling. Evid Comp Alternat Med2012;2012:1155-63.
Vergouwen MD, Schut ES, Troost D, van de Beek D Diffuse cerebral intravascular coagulation and cerebral infarction in Pneumococcal meningitis
. Neurocrit Care 2010;13:217-27.
Ugwu Okechukwu PC, Nwodo Okwesili FC, Joshua Parker E, Abubakar B, Ossai Emmanuel C, Odo Christian E Phytochemical and acute toxicity studies of moringa oleifera ethanol leaf extract. Int J Life Sci Bt Pharm2013;2:66-71.
Chesbrough M District laboratory practice in tropical countries. Part 2; Cambridge, United Kingdom: Cambridge University Press, 2018 . p. 62-71, 116-29.
National Research Council. Guide to the care and use of laboratory animals resources. DHHS; 2018. p. 86-23.
Morrison SJ, Scadden DT The bone marrow niche for haematopoietic stem cells. Nature 2014;505:327-34.
Thompson EC Focus issue: Structure and function of lymphoid tissues. ScienceDirect 2012;33:255.
Winter O, Moser K, Mohr E, Zotos D, Kaminski H, Szyska M, et al
. Megakaryocytes constitute a functional component of a plasma cell niche in the bone marrow. Blood 2010;116:1867-75.
Sohail I, Ghosh S, Mukundan S, Zelewski S, Khan MN Role of inflammatory risk factors in the pathogenesis of Streptococcus pneumoniae
. Front Immunol 2018;9:2275.
Luca DC Treatment related changes in bone marrow. Available from: https://www.pathologyoutlines.com/topic/bonemarrowtreatmentrelated.html. [Last accessed on 2019 Dec 31].
Ramaiah L, Bounous DI, Elmore SA Hematopoietic system, Haschek and Rousseaux’s handbook of toxicologic pathology. 3rd ed. Vol. 3; London: Academic Press, 2013. p. 1863-933.
Cunin P, Bouslama R, Machlus KR, Martínez-Bonet M, Lee PY, Wactor A, et al
. Megakaryocyte emperipolesis mediates membrane transfer from intracytoplasmic neutrophils to platelets. eLife 2019. Available from: https://doi.org/10.7554/eLife.44031.001
Di Buduo CA, Alberelli MA, Glembostky AC, Podda G, Lev PR, Cattaneo M, et al
. Abnormal proplatelet formation and emperipolesis in cultured human megakaryocytes from gray platelet syndrome patients. Sci Rep 2016;6:23213.
[Plate A], [Plate B], [Plate C], [Plate D1], [Plate D2], [Plate E1], [Plate E2], [Plate F1], [Plate F2]