evaluation of pain alleviating, anti-inflammatory and …€¦ · evaluation of pain alleviating,...
TRANSCRIPT
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EVALUATION OF PAIN ALLEVIATING, ANTI-INFLAMMATORY AND ANTI-
RHEUMATIC PROPERTIES OF SELECTED KENYAN MEDICINAL PLANTS
A proposal submitted in fulfillment of requirements for Doctor of Philosophy degree of
University of Nairobi, Physiology.
Stanley Nderitu Wambugu, BVM, MSc
Department of Veterinary Anatomy and Physiology
Signature…………………………. Date……………
iii
This proposal has been submitted with our approval as university supervisors.
1. Prof. Titus I. Kanui, PhD.
Signature…………………………... Date…….
Department of Vet. Anatomy and Physiology,
2. Dr. Stephen G. Kiama, PhD.
Signature…………………………... Date…….
Department of Vet. Anat. and Physiology,
Faculty of Veterinary Medicine, U.o.N.
3. Dr. Peter Mbaabu Mathiu, PhD,
Signature…………………………... Date…….
Department of Vet. Anat. and Physiology,
Faculty of Veterinary Medicine, U.o.N.
4. Dr. Daniel W. Gakuya, PhD.
Signature…………………………... Date…….
Department of Clinical Studies,
All of the Faculty of Veterinary Medicine, University of Nairobi
Date of submission…………………………………………………………..
iv
TABLE OF CONTENTS
CHAPTER ONE ............................................................................................................................. 1
1. INTRODUCTION ................................................................................................................... 1
CHAPTER TWO ............................................................................................................................ 2
2. Literature review ...................................................................................................................... 2
2.2 Objectives ....................................................................................................................... 4
2.2.1 Specific objectives: ..................................................................................................... 4
CHAPTER THREE ........................................................................................................................ 5
3.0 MATERIALS AND METHODS ........................................................................................ 5
3.1 Ethnobotanical survey .................................................................................................... 5
3.2 Sample preparation ......................................................................................................... 6
3.3 Evaluation of the anti-nociceptive activity in the Tail-flick test ..................................... 6
3.4 The Evaluation of the Analgesic and Anti-Inflammatory Activity in the Formalin Test 7
3.5 Evaluation of Anti-Arthritic Effect in Adjuvant-Induced Arthritis in Rats ..................... 8
3.6 Induction of RA Rats Using the Collagen Induced Arthritis (CIA) ................................ 9
3.6.1 Macroscopic scoring system: .................................................................................... 10
3.6.2 Hind paw diameter .................................................................................................... 10
3.6.3 Wetting with acetone ................................................................................................. 11
3.6.4 Biochemical assay of pro-inflammatory and anti-inflammatory cytokines .............. 11
3.7 Statistical analysis ......................................................................................................... 12
References ..................................................................................................................................... 12
Budget .............................................................................................................................................. i
Work plan ........................................................................................................................................ ii
Appendix 1: Data acquisition questionnaire .................................................................................. iii
Appendix 2: Histological grading of arthritis ................................................................................ vi
1
CHAPTER ONE
1. INTRODUCTION
Rheumatoid arthritis (RA) is a complex chronic inflammatory condition with a systemic
autoimmune disorder that manifests with pain, inflammation and tissue damage in joints,
together with anemia. The disease has idiopathic etiology and a rather unusual pathogenesis
which makes it difficulty to identify effective therapies (Wood, 2004; Hultqvist et al., 2006;
Lindquist 2002). Osteoarthritis (OA) on the other hand is a degenerative joint disease
characterized by damage to the articular cartilage, changes in subchondral and marginal bone,
synovitis and capsular thickening, usually affecting the weight bearing joints (Bendele, 2001;
Schaller et al., 2005).
Conventionally, the treatments of intense and unrelenting pain, such as that resulting from
rheumatoid arthritis, joint pain, neuropathic pain or injury is based on opioids, non-steroidal anti-
inflammatory drugs (NSAIDs) and drugs inhibiting inflammatory cytokines despite their well
documented side effects and prohibitive cost. On the contrary, natural products offer a more
accessible and cheaper alternative, and they have even contributed enormously to the
development of current therapeutic agents (Setty and Sigal 2005; Lipsy and Tao, 2007).
However, most of the commonly used herbal remedies used by the Kenyan TMP have not been
systematically validated. In addition, there’s a rapid disappearance of traditional herbal
knowledge and increased biodiversity degradation all threatening the loss of our economically
valuable cultural heritage (Srithi et al., 2009). All these calls upon for conscious and concerted
efforts to collect, document and scientifically validate medicinal plants use in our communities.
The commonly used medicinal plant preparations for the management of OA and RA are usually
combined plant extracts, and indeed this may the future direction in the development of
2
efficacious, safe and cost effective herbal remedies world over (Lee 2000; Chrubasik et al.,
2007; Park et al., 2007). These herbal remedies have actually been recognized as effective, safe
and cost effective agents and indeed several herbal based traditional medicinal preparations are
patented as phytopharmaceutical products (Darshan and Doreswamy 2004). In Kenya, several
medicinal preparations are used in the management of osteoarthritis and rheumatoid arthritis and
the objective of this study will be to investigate the analgesic, anti-inflammatory and anti-
rheumatoid properties of selected medicinal plant preparations/extracts in Machakos/Makueni
region of Kenya.
CHAPTER TWO
2. Literature review
Osteoarthritis (OA) and rheumatoid arthritis (RA) are among the most common joint ailments
and a leading cause of irreversible joint deformities and functional impairment (Wood, 2004).
Moreover, these conditions often lead to both long term morbidity and early mortality (Kelly et
al., 1997; Doran et al., 2002) and the economic burdens associated with their management are
devastating. RA affects 1-2% of the population is more common in women than in men while
osteoarthritis is a common problems affecting over 60% of all people by the age of 50 years
(Wood 2004; Schaller et al., 2005; Lawrence et al., 2008).
Over the years, there has been a variety of exciting new therapies for the management of OA and
RA with improved patient responses based on clinical signs and symptoms reported (Wood
2004; Setty and Sigal 2005; Caporali et 2008). However, all these therapies have not been
without devastating side effects, some of which are life threatening but remain in use only by the
terminally ill patients where the safety vs. severity of the condition is the driving force towards
their use. For instance, the most commonly prescribed DMARDs are associated with a range of
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side effects related to liver and bone marrow toxicity (methotrexate, sulfasalazine, leflunomide,
azathioprine, gold salts, D-penicillamine), renal toxicity (cyclosporine A, parenteral gold salts,
D-penicillamine), pneumonitis (methotrexate), allergic skin reactions (gold compounds,
sulfasalazine), autoimmunity (D-penicillamine, sulfasalazine, minocycline) and infections
(azathioprine, cyclosporine A) (Mikuls, 2003; Fleischman et al., 2004; Woods 2004; Majithia
and Geraci, 2007; Caporali et al., 2008). TNF and interleukin antagonists have been implicated
with serious infections, malignancies, heart failure, chronic demyelinating disorders, lupus-like
autoimmune disease, vasculitis, liver abnormalities and hematological abnormalities (Francis
1999; Mohan et al., 2001; Brown et al., 2002; Kroesen et al., 2003; Wood 2004).
Like many other chronic diseases RA and OA are associated a high usage of herbal medicines
(Resch et al., 1997; Lipsy and Tao, 2007; Setty and Sigal 2005). According to World Heath
Organization statistics, more than 80 percent of the population in developing countries continues
to rely on traditional medicine and indigenous knowledge to meet their health needs (Nanyingi et
al., 2008). Owing to the fact that traditional medicine is accessible, affordable, culturally
acceptable, socially sanctioned, and easy to prepare with little or no side effects, most people
prefer it to the exorbitantly priced health care services conventional medicine and practitioners
offer, not to mention the hazardous side effects of these medications and their limitations in the
domain of holistic health, especially in economically impoverished African societies.
Thus, there is a growing need for safe and effective traditional medicine therapies, an important
tool to increase access to health care particularly in poor and marginalized populations Several
plants species are used in the management of these conditions and but very few have been
systemically investigated for their efficacy. This study hopes to come up with a valid anti-
arthritic and anti-rheumatoid herbal formula.
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2.2 Objectives
The general objective will be to investigate the analgesic, anti-inflammatory and anti-rheumatoid
properties of selected medicinal plants used by some Kenyan TMP.
2.2.1 Specific objectives:
1. To analyze the diversity of medicinal plants used in the management of RA by different
TMP in the selected localities (Machakos/Makueni area). The entire method of
preparation, extraction and formulation will also be analyzed and documented.
2. To evaluate the pain alleviating properties of the various plants parts/extracts and
preparations using the tail flick and the formalin tests. Analgesic potency will also be
analyzed in the formalin test.
3. To investigate the anti-arthritic properties of selected plant extract/preparation in the
Complete Freund’s Adjuvant (CFA) arthritis model in rats.
4. To evaluate the anti-rheumatoid properties of the plant extract/preparation in the
Collagen-Induced Arthritis model in rats.
5. To evaluate the immunosuppressive effects of the plant extract/preparation in rats.
6. To study the histopathological effects of the antiiarthritic and anti-rheumatoid plant
extract/preparation.
7. Perform separation of the most active anti-arthritic/rheumatoid extracts/preparation to
elucidate on the biochemical composition of the most active ingredients.
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2.3 Hypothesis
The commonly used herbal medicine preparation and plant extracts have significant pain
relieving, anti-arthritic and anti-rheumatoid properties and are safe for medicinal usage.
2.4 Justification
Joint pain and rheumatoid arthritis are difficulty and expensive conditions to manage especially
in economically impoverished communities where provision of primary healthcare is inexistent
or inadequate. There is also a great demand for new anti-rheumatic agents that are able to act on
chemical mediators of inflammation, but have fewer toxic effects and cost less than the current
treatments that block the actions of TNF-α, IL-1/6 or those of the Disease Modifying Anti-
Rheumatic Drugs (DMARDs) (Majithia and Geraci, 2007).
The therefore aims to identify and verify the efficacy of locally used medicinal plant
formulations used in the management of the two joint conditions. Individual plant extracts and
the medicinal preparation will also be compared for relative potency. The study hopes to come
validate the hypothesis that the commonly used herbal preparation/plant extracts are safe,
cheaper and effective in the management of rheumatoid arthritis and osteoarthritis.
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 Ethnobotanical survey
An ethnobotanical survey will be carried out in Machakos and Makueni districts to identify the
plants and procedures involved in the making of the medicinal preparation used in the
management of RA/OA. Field visits will be done and semi-structured questionnaires
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administered to registered traditional herbal practitioners (TMP)to obtain data regarding the
diversity of plants used, methods of preparation and formulation, diagnosis, dosage, treatment
outcomes and prognostic indicators, recurrence as well as patient satisfaction. A sample
questionnaire is shown on appendix 1. Data obtained from the questionnaire will be analyzed
using the appropriate methods and the commonly used plants collected for taxonomic
identification. Voucher specimens will be deposited at the national museums of Kenya
herbarium/UoN herbarium.
3.2 Sample preparation
The plants of interest will be harvested and dried under the shade. They will be processed into
powder form before extraction, which will be done in accordance to the methods used by the
TMP. For comparative purposes, methanolic, chloroform and metnanolic: diclorolomethanolic
extracts will also be prepared by cold extraction. Basically the dry powdered plant material will
be macerated in the solvent extractor for 3 days at room temperature and the supernatant filtered
in whatman no.1 paper. The residue from the filtration will also be extracted again twice using
the same procedure. The filtrates obtained will be combined and then evaporated to dryness
under reduced pressure to obtain a lyophilized extract. The percent yield will be calculated as per
the equation % yield = Wcrude extract/Wdried plant × 100 where Wcrude extract = weight of
crude extract and Wdried plant = weight of dried plant material (Phrompittayarata et al., 2007).
3.3 Evaluation of the anti-nociceptive activity in the Tail-flick test
The anti-nociceptive activity of the plant extract/preparation will be assessed using the tail flick
test. This is a phasic pain test that measures tail flick responses to radiant heat stimulation, a
spinally integrated nociceptive reflex (Le Bars et al., 2001). The test is simple to perform,
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displays minimal inter-animal variability and allows for repeated testing without conditioning
effects, making it a useful tool for screening analgesic drugs (Langerman et al., 1995; Le Bars et
al., 2001).
The mice will be kept in cages in a well ventilated drought free house with a 12 hour light-dark
cycle and provided with food and water ad libitum. The test room will be maintained at equal
ambient temperature to that of the animal house. A randomized block design will be used based
on the sex of the animals. The test stimulus will be preset at a temperature that rises from 35ºC to
55ºC in ten seconds. The heating will be terminated manually when a tail flick response occurs.
Tail flick latency will be scored and a cut off time of 40 sec will be adopted. Testing will be done
at -10, 0, 0.5, 1, 2, 3, 4, 5 hours. The average of -10 and 0 (pretreatment scores) will be used as
the base line (control) and test material will be administered at time 0. The extract will be
administered i.p at two predetermined dosages with a saline treated control group. The person
scoring the nocifensive behavior will not be aware of what has been injected into the animals.
3.4 The Evaluation of the Analgesic and Anti-Inflammatory Activity in the Formalin Test
The method will be adapted from that described by Rosland et al (1990). In this tonic pain test, a
20 µL of 0.5% formalin solution will be injected into the dorsal surface of the hind paw in mice.
Scoring will be based on the duration of time spent in nocifensive behavior for 60 min in blocks
of five minutes. In this test, the nocifensive behavior will be defined as licking and biting of the
formalin injected paw. The mean duration of time spent in pain in the first 10 minutes will be
taken as the pain response in the first phase of the formalin test, while the late phase will be
taken as between 20 and 45 minutes after injection of the formalin. The observer will not be
aware of what has been administered in the animals.
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To evaluate for the antinociceptive potency of the antinociceptive activity of the plant
extracts/preparation, the effects will be compared with those of morphine, aspirin and
dexamethasone. Treatment (with the plant material, vehicle, morphine, naloxone, aspirin and
dexamethasone) will be done i.p at two predetermined dosages 30 min before the formalin
injection. The percentage inhibition of licking/biting will be calculated the formula:
[(C−T)/C] × 100; where C represents the vehicle treated control group value for each phase and
T represents the treated group value for each phase (Dongmo et al., 2005).
3.5 Evaluation of Anti-Arthritic Effect in Adjuvant-Induced Arthritis in Rats
Experimental arthritis will be induced in rats according to the method described by Jia et al
(2003). In this chronic pain model, the stimulus is inflammatory reaction in the joints caused by
the injected material and involves intradermal injection of Mycobacterium butyricum with
Freund’s adjuvant into the tails (Bendele, 2001; Le Bars et al., 2001; Yu et al., 2002). Rat
adjuvant induced arthritis is widely used experimental model of polyarthritis, periarthritis and
periosteitis (Mendele, 2001; Yu et al., 2002; Jia et al., 2003) and the polyarthritis induced is
similar to various inflammatory conditions. Furthermore, results from this test are predictive of
analgesic and anti-inflammatory activity of the test substance (Bendele, 2001; Cho et al., 2008).
The right footpad of each rat will be injected S.Q with 0.1 ml of CFA (Sigma). The plant
material will be administered i.p at three predetermined dose levels, distilled water, and
acetylsalicylic acid at 10 mg/kg will be given daily to the treatment groups, control group, and
reference group, respectively, starting at 8 days after CFA injection for 14 consecutive days. The
edema (paw perimeter) of the left and right hind paws will be evaluated at 2 h, 8, 12, 16, and 24
days post-injection of CFA. Experiments terminated at day 24. The edema inhibition will be
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calculated as follows: Inhibition rate (%) = Vc - Vt/Vc × 100, where Vc is the edema value of
the control group; and Vt is the edema value of treated group.
3.6 Induction of RA Rats Using the Collagen Induced Arthritis (CIA)
Adult (10-15 wk) male Sprague Dawley rats will be used in these experiments due to their
susceptibility to collagen induced arthritis (CIA), the most commonly used animal models of
rheumatoid arthritis which involves both humoral and cell-mediated immune components
(Trentham, 1982; Cho et al., 2007; Bendele, 2001). The arthritis ensues when rats are immunized
against homologous or heterologous type II collagen, and is characterized by marked bone
destruction associated with deposition of immune complexes on articular surfaces, bone
resorption, periosteal proliferation, synovitis and periarticular inflammation (Mendele 2001;
Maruyama et al., 2006). The rats will be housed in groups of 10 and maintained at a temperature
of 21 ± 2°C on a 12-hour light/dark cycle (7:00 am to 7:00 pm) and provided with food and
water ad libitum.
Collagen II from bovine articular cartilage will be dissolved overnight at 4◦C in 0.1M acetic acid
at a concentration of 2.5mg/mL. The solution will be emulsified with 1.2 times volume of
Complete Freund’s Adjuvant (CFA), and 100 µL of the emulsion administered subcutaneously at
the base of the tail of the rats for immunization on day 1 (see figure 1). Booster injection of 100
µL of the emulsion will be given on day 15. The rats will be given the plant extract/preparation
orally by galvaging at predetermined dosages from day 0 to 21, 5 days per week. Control rats
will be given an equal volume of 2.5% DMSO solution. Their weight and paw diameters will be
measured 2 days each week from day 0 to 39.
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Fig.1: Schematic diagram of the main activities during the analysis of RA in CIA rats.
Scoring will be done by monitored by assessment of several different parameters at various time
points before and after induction of arthritis.
3.6.1 Macroscopic scoring system:
The extent of paw swelling and edema shall be scored as the method of Paola and Cuzzocrea
(2008), with a score ranging from 0 to 3 (0, no swelling; 1, slight swelling and/or erythema; 2,
pronounced edematous swelling and 3, severe arthritis and joint rigidity). Each limb shall be
graded, thus allowing a maximum score of 12. The arthritic score for each mouse will be the sum
of the scores of all four paws (Paola and Cuzzocrea, 2008).
3.6.2 Hind paw diameter
To investigate the effects of treatments on edema in arthritic joints, tibiotarsal joint
circumferences will be measured using a tape measure and the change in paw size after
treatments expressed as percent change in paw diameter (Chillingworth and Donaldson 2003).
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3.6.3 Wetting with acetone
This test is used to measure cold allodynia in animal models of pain (Kim et al., 2008). Acetone
will be applied five times on the dorsal surface of the hind paw at intervals of more than 2
minutes at room temperature. Brisk withdrawals will be measured and the data expressed as paw
withdrawal latency (PWL), which is the number of responses divided by number of trials
expressed as a percentage (Kim, 2008). PWL = Number of responses/ Number of trials * 100.
3.6.4 Biochemical assay of pro-inflammatory and anti-inflammatory cytokines
Cytokines, a diverse group of polypeptides that are generally associated with inflammation,
immune activation, and cell differentiation or death, include Interleukins (IL), Interferon’s (IFN),
tumor necrosis factors (TNF), Chemokines and growth factors (Dinarello, 2000; Wong et al.,
2008). Moreover, the balance between the effects of pro-inflammatory and anti-inflammatory
cytokines thought to be the key determinant in the outcomes of an inflammatory reaction as well
as therapeutic efficacies of agents (Wong et al., 2008).in this study, The levels of TNF-ά, IL-1 β
(pro-inflammatory) and INF-ά (anti-inflammatory) will be analyzed from blood samples
collected at different levels of RA (as shown in figure 1) using a rat cytokine ELISA kit (BioPlex
cytokine kit, BD, UK) in accordance with the manufacturers instructions. Rat paw tissue
exudates collected immediately after sacrificing the animals will also be assayed.
3.6.5 Histopathological grading of Rheumatoid Arthritis
This will be based on the three main features of chronic arthritis (Chillingworth and Donaldson
2003; Krenn et al., 2006). These are enlargement of synovial lining, cellular density of synovial
stroma and leukocyte infiltration. Each will be assigned a score of 0-3, with 0 being no change
and 3 the high grade synovitis. The arthritic score for each rat will be the sum of the scores of all
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four paws. Hind ankle joints will be dissected on day 35, fixed in 10% neutral buffer formalin,
decalcified in 10% formic acid-formalin, and embedded in paraffin. The hind ankle joints will be
sectioned at 4 µm and stained with haematoxylin and eosin and blind scored for arthritis as
shown in appendix 2.
3.7 Statistical analysis
All the data will be expressed as mean ± standard error of the mean (SEM) and statistically
compared using one-way analysis of variance (ANOVA). Post hoc comparisons of the
differences between the drug-treated groups and controls will be performed using Dunnett’s t-
test and P values < 0.05 will be considered significant.
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Budget
ITEM Unit Cost
(KShs)
Quantity Total (KShs)
Tuition fees 150, 000 per yr 3 years 450,000
Ethnobotanical survey 45,000 2 90,000
Mice 100 1000 100,000
Dichloromethane 3000 per 5 lits 20 lits 12,000
Dimethyl-sulphoxide 3000 per 5 lits 20 lits 12,000
Methanol 3000 per 5 lits 20 lits 12,000
Chloroform 3000 per 5 lits 20 12,000
Collagen Induced Arthritis
(CIA) kit
50,000 1 50,000
Cytokine Assay kit 68,000 1 68,000
Harvesting of plants 25,000 2 50,000
Transport costs 20,000 5 100,000
Data storage, handling and
analysis
30, 000
Na-pentobarbitone100ml 5000 1 5000
Syringes, Needles, slides,
cover slips and other
consumables
30,000
Sub-total 1, 021,000
10% Contingency 102,100
GRAND TOTAL 1,123,100
ii
Work plan
Activity Year 1 Year 2 Year 3
Month 1- 4 5-8 9-12 1-4 5-8 9-12 1-4 5-8 9-12
Literature review, PhD
registration and
ethnobotanical survey
Plant collection and
preparation of extracts
Antinociceptive and
analgesic testing
Induction of CIA in
mice and testing for
anti-rheumatic
properties of herbal
extracts
In-vitro analysis and
histological analysis of
arthritis
Data entry and cleaning
Data analysis
Determination of
dosage
Standardization
Thesis writing,
submission
Dissemination of
research
findings/publication
iii
Appendix 1: Data acquisition questionnaire
Data acquisition questionnaire on plant extract/preparation usage in OA and RA
QUESTIONNAIRE
PART 1: RESPONDENTS DETAILS
Name............................................................... Sex....M/F Age...........Years.
Occupation........................................................
Registered ………….. (Yes/No). If yes, note the registration number/details……………..
Level of education........................................
Location/Residence..........................................................................................................................
Efficacy/Availability/Toxicity Data
Name and part(s) of Plant (Local name)......................................................used in OA........ RA.....
Availability of the plant (abundant/scarce/endangered) .................................................................
Preparation method(s)..........................................................................................
Formulation used (mixed with)………………………………………………… in OA…..RA…..
Administration form (s)..........................................................................................
Route(s) of application ..........................................................................................
Diagnosis OA ………………………………………… RA ……………………………………
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Approximate dosage..........................................................................................
Response of Patient Good.........................Fair........................ Poor.........................
Duration of response: Minutes....... Hours.......... Days …….. Months…… Years……..
Complication……………………………………………………………………………….
Shelf life of the formulation …………………………………………………………….
Treatment outcomes and recurrence ……………………………………………………
Prognostic indicators …………………………………….………………………………
Patient satisfaction………………………………………………………….……………..
Proportion of initial case vs. cases initially reported (and treated) else where ……….. …
………………………… ………………………………………………………….….
PART 2: RESPONDENTS CONSENT AGREEMENT
I.......................................................................................Hereby agree to participate in this study
with my full consent and conscious and declare that to the best of my Knowledge the information
that I have provided is true, accurate and complete.
Signature/Thumb print...........................................Date.............
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PART 3: RESEARCHER'S DECLARATION
1. The following research will be undertaken with respect to the indigenous knowledge and
intellectual proprietary of the herbal practioners.
2. We will at no given time initiate or conduct practices that are deemed to obtain
information from the respondents by intimidation, coercion or false pretence.
3. The respondents will be informed of the intended project prior to questionnaire
administration and in confidential to eliminate any degree of conspiracy.
4. We will be no under any obligation to edit or tamper the information provided by the
respondents.
5. The information collected will be used for the described research purpose and not any
undisclosed intentions.
Signatory Researchers:
1. Dr. Wambugu S.N..................................... Prof. Kanui T.I..........................
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Appendix 2: Histological grading of arthritis
1. Enlargement of the synovial lining cell layer (at X 200).
0 points The lining cells form one layer.
1 points The lining cells form 2-3 layers.
2 points The lining cells form 5-4 layers, few multinucleated cells may occur.
3 points The lining cells form more than 5 layers, lining might be ulcerated and
multinucleated cells may occur.
2. Density of the resident cells (at X200)
0 points The synovial stroma shows normal cellularity
1 point The cellularity is slightly increased
2 points The cellularity is moderately increased, multinucleated cells might occur
3 points
The cellularity is greatly increased, multinucleated giant cells, pannus
formation and rheumatoid granulomas might occur
3. Inflammatory infiltrate (at X400)
0 points No inflammatory infiltrate
1 point Few mostly perivascular situated lymphocytes or plasma cells
2 point Numerous lymphocytes or plasma cells, sometimes forming follicle-like aggregates
3 points Dense band-like inflammatory infiltrate or numerous large follicle-like aggregates
Sum 0 or 1 = No synovitis
Sum 2–4 = Low-grade synovitis
Sum 5–9 = High-grade synovitis