الرحمن الله الرحمن بسم الله بسمالرحيمالرحيم

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العظيم الله العظيم صدق الله صدق((3232)البقرة:)البقرة:

الرحمن الله الرحمن بسم الله بسمالرحيمالرحيم

ل�م� ل�ن�ا ”” ل�م� ل�ن�ا قالوا سبحانك ال ع قالوا سبحانك ال ع�ن�ت� ن�ك� أ ت�ن�ا إ ا ع�ل�م� �ن�ت� إال م� ن�ك� أ ت�ن�ا إ ا ع�ل�م� إال م�

�كيم كيم�ال�ع�ليم� ال�ح� ““ال�ع�ليم� ال�ح�

العظيم الله العظيم صدق الله صدق((3232)البقرة:)البقرة:

BYKHALID M. GHARIB MDLECTURER OF DERMATOLOGY AND VENEREOLOGY ZAGAZIG UNIVERISTY

INTRODUCTIONINTRODUCTION

ANDROGENETIC ALOPECIAAndrogenetic alopecia (AGA) also referred to as

male-pattern hair loss, common baldness or female-pattern hair loss, is a slowly progressive form of alopecia that begins after puberty.

It affects at least 50% of men by the age of 50 years, and up to 70% of all males in later life.

AGA is characterized by androgen dependent, progressive, patterned hair loss from the scalp (Zhao and Hantash, 2011).

The pathogenesis of AGA is poorly understood. It is

assumed that the genetically predisposed hair

follicles are the target for androgen-stimulated hair

follicle miniaturization, leading to gradual

replacement of terminal hairs by barely visible,

depigmented vellus hairs.

The miniaturization process occurs over many

years during repeated hair cycles with shortened

anagen phase and hair follicle reduction in size and

depth (Trüeb , 2002).

Hair follicle is the main source of multipoint stem

cells in the skin. These stem cells reside in a

quiescent niche in bulge region near the insertion of

arector pill muscle. Bulge cells generate all the

epithelium lineages within the follicle and their

selective destruction leads to loss of hair follicle.

Also, bulge cells give rise to progenitor population

called secondary germ cells that reside adjacent to

bulge during telogen and produce the new hair shaft

at anagen onset (Zhang et al., 2009).

STEM CELL MARKERS

Recently, due to discovery of cell surface markers, it has become possible to characterize the hair follicle stem cells.

Cytokeratin15 is the best marker for bulge epithelial stem cells in human hair.

Other markers as Nestin, p63 and CD34 allow assessment of stem and progenitor cell populations in human scalp.

Several studies, reported loss of CK15+ cells in some

scarring alopecia, implying invovement of bulge

region in its pathogenesis (Tsuboi et al., 2012).

AGA represents the most common type of non

scarring alopecia; however permanent alopecia may

occur in long standing cases. This irreversibility of

the ability to grow hair again may give rise to the

possibility of stem cell invovement in this type of

alopecia (Al-Refu , 2012).

Recently, few studies have been investigating the status

of the hair follicle stem cell compartment in AGA.

Some studies suggested that compromising integrity of

follicular bulge area or sebaceous gland may play a role

(Lee and Lee, 2012).

While others suggested diminished conversion of hair

follicle stem cells to progenitor cells. whether these data

will assess the role of hair follicle stem cells in AGA is still

unknown.

AIM OF THE AIM OF THE WORKWORK

The aim of the present study was The aim of the present study was

to investigate the possible role of to investigate the possible role of

hair follicle stem and progenitor hair follicle stem and progenitor

cells in the pathogenesis of cells in the pathogenesis of

androgenetic alopeciaandrogenetic alopecia.

PATIENTS&METHODSPATIENTS&METHODS

This study has been conducted on 20 patients with AGA selected from the outpatient clinics of the Dermatology Department of Zagazig University Hospitals during the period from January 2011 to December 2012.

They were 17 males and 3 females with their ages ranging from 20 to 29 years with a mean of 24.05±1.6.

Diagnosis of AGA was clinically established in males by the characteristic distribution of frontal and vertex hair and in females by the Christmas tree pattern of diffuse hair loss at middle hairline.

INCLUSION CRITERIAINCLUSION CRITERIA:: Patients more than 20 years old, both sex.

Patients diagnosed as male AGA, type III to VI

using Hamilton –Norwood classification, and as

female pattern hair loss type I-III using Ludwig

classification.

Active hair loss within last 12 months.

Willing to sign informed consent.

Norwood -Hamilton scale Norwood -Hamilton scale Ludwig scaleLudwig scale

I II III Stage

EXCLUSION CRITERIAEXCLUSION CRITERIA:

Pregnancy

Malignancy or other systemic disease that might

interfere with diagnosis as collagen diseases and

chronic thyroiditis.

Unconfirmed diagnosis clinically or pathologically.

Global scalp hair thinning including occipital areas.

IMMUNOHISTOCHEMICAL STUDYFour mm punch biopsy specimens were

obtained from each patient from both frontal

affected area of scalp and the occipital skin that

served as a control.

The specimens were fixed in 10% neutral

buffered formalin and processed for paraffin

embedding.

Serial 4 µm sections from paraffin blocks were stained with:

Hx.& E. stain for histopathological diagnosis.

Immunohistochemical staining using the Streptavidin-Biotin

immunoperoxidase technique for the detection of:

CK15 expression using CK15 Ab-1 mouse monoclonal AB.CK15 expression using CK15 Ab-1 mouse monoclonal AB.

CD34 expression using CD34 mouse monoclonal AB (1:800).CD34 expression using CD34 mouse monoclonal AB (1:800).

P63 expression using P63 Ab-1 mouse monoclonal antibody P63 expression using P63 Ab-1 mouse monoclonal antibody

(Wallace and Smoller, 1997).(Wallace and Smoller, 1997).

EVALUATION OF STAINING RESULTSEVALUATION OF STAINING RESULTS:

CK15 positive staining was noted by ascertaining CK15 expression in cytoplasm. Any nuclear stain was considered background artifact.

P63 positive expression was noted by nuclear localization. CD34 expression was noted when uniform/circumferential staining expression as seen in occipital skin was not observed (Hoang et al., 2009).

Positive slides showed brown colored precipitate. The percentage of positive cells was calculated in 100 cells / 4 HPF.

CRITERIA FOR GRADING STAINED SECTIONSCRITERIA FOR GRADING STAINED SECTIONS

Negative (-): if <5 % positive cells.

Weakly positive (+): if 5% - 25% positive cells.

Moderately positive (++): if >25-50% positive cells.

Strongly positive (+++): if >50% positive cells

(Fiuraskova et al., 2005).

STAISTICAL ANALYSIS

The data were tabulated and statistically analyzed

using the SPSS for Windows 10.0 statistical package

program. Paired data of qualitative variable were

estimated by McNemar,s chi-square test (X2) and

Wilcoxon signed rank test `(Dean et al., 1994)

RESULTSRESULTS

This study was conducted on 20 AGA patients.

Seventeen patients (80%) were males and three patients

(15%) were females. Their ages ranged between 20 and

29 years with a mean of 24.05± 1.6.

Classification of males according to Norwood Hamilton

scale showed that two patients (10%) were of type IV, 3

patients (15%) of type V, 4 patients (20%) of type Va and

8 patients (40%) of type VI AGA.

Using Ludwig's classification in females, 1 patient (5%)

was of type II and 2 patients (10%) were of type III AGA.

The disease duration ranged between 1-6 years with a

mean of 3.175 ± 2.014 years. No family history of a

similar condition was detected.

All patients had received previous therapy, 85% received

topical minoxidil therapy while 15% received intralesional

mesotherapy .

Table (1): Demographic data and clinical characteristics

Immunohistochemical examination of scalp biopsies

using CK15 antibody showed mild positive expression of

CK15 in both frontal and occipital skin in all 20 AGA

patients (100%).

CK15 was expressed in the bulge region and in the

peripheral layer of the outer root sheath, at the level of the

isthmus.

In frontal skin, CK15 was expressed in the bulge region in

95% (19 patients) and in the peripheral layer of ORS

in100% (20 patients) compared to 100% in both the bulge

region and ORS in occipital skin.

Table (2): Summary of CK15 positive expression in frontal versus occipital skin in 20 AGA patients.

Table (3): Staining degree of CK15 in frontal versus occipital skin

Specifically, the CK15

immunostaining extended

from the entrance of the

sebaceous gland duct

down to the insertion site

of the arrector pili muscle.

This CK15 staining was

maintained throughout the

different phases of the

hair follicle cycle.

Figure (1): CK 15 positivity of frontal hair follicle within bulge region and ORS (X400).

1

Fig (2): CK15 positivity within occipital hair follicle (X200).

Fig(3): CK 15 positivity of frontal hair follicle of same patient at the outermost cell layer of

ORS, at the level of isthmus( X400) .

Figure (4): CK15 positivity within frontal hair follicle at level of sebaceous glands, (X200).

CD34 ANTIBODY`

Immunohistochemical examination using CD34

antibody showed positive expression in 30%

(6 patients) of frontal scalp biopsies, compared to

85% (17 patients) in occipital biopsies with a highly

statistically significant difference, (P<0.001) .

Table (4): Comparison of CD34 expression in frontal versus occipital skin.

A highly statistically significant difference was also

detected in CD34 staining intensity in frontal skin as

compared to occipital (P<0.005).

In frontal skin, weak positivity was detected in 20% (4

patients), moderate positivity in 5% (1 patient) and

strong positivity in 5% (1 patient). While in occipital

skin, 65% (13 patients) showed weak positivity, 5% (1

patient) showed moderate positivity and 15% (3

patients) showed strong positivity .

Table (5): Staining degree of CD34 in frontal versus occipital skin .

The CD34 immunoreactivity was detected only

below the bulge zone, at the most peripheral

layer of ORS, in the transient portion of follicle

below the isthmus and above the matrix cells.

Thus CD34 and CK15 antibodies recognize

different types of cells or cells at different stages

of differentiation.

Figure (5): CD34 positive expression in control occipital scalp (X200).

Figure (6): CD34 negative expression in frontal scalp follicles while positive expression in blood vessels (X200).

Figure (7): CD 34 positive expression in control occipital scalp (X200).

Figure (8): CD34 negative expression in frontal scalp of same patient (X400).

CD34 immunoreactivity was not detected in

catagen or telogen hair follicles. Only anagen

hair follicles showed CD34 immunoreactivity.

Other skin structures that stained with CD34

antibody were the endothelial cells and

perifollicular spindle shaped cells .

Figure (9): CD34 negative expression in telogen hair (X200).

P63 ANTIBODYImmunohistochemical exam. using P63 antibody

showed positive expression in 85% (17 patients) of frontal scalp biopsies, compared to 100% (20 patients) in occipital biopsies with no statistically significant difference, (P>0.08).

The nuclear staining for P63 was detected in all hair follicle epithelial structures. However, comparison of P63 staining intensity in frontal versus occipital skin showed a highly statistically significant difference (P=0.005).

Table (6): Comparison of p 63 expression in frontal versus occipital skin.

In frontal skin, weak positivity was detected in 40% (8 patients), moderate positivity in 30% (6 patients) and strong positivity in 15% (3 patients).

While in occipital skin, 45% (9 patients) showed weak positivity, 25% (5 patients) showed moderate positivity and 30% (6 patients) showed strong positivity.

Thus higher expression of P63 is detected in occipital skin as compared to the affected frontal skin.

Table (7): Staining degree of P63 in frontal versus occipital skin.

Figure (10): p63 strong positive expression in occipital area (X200).

Figure (11): p63 positive expression in frontal area (X200).

Figure (12): p63 strong positivity in occipital area (X400).

Figure (13): p63 strong positivity in occipital area (X400).

Figure (14): p63 positivity in telogen germinal center in frontal area (X400).

CONCLUSIONCONCLUSION

& &

RECOMMENDATIONSRECOMMENDATIONS

In conclusion, although the pathogenesis of In conclusion, although the pathogenesis of

AGA is still not understood, this study confirms AGA is still not understood, this study confirms

that the follicular stem cells in the bulge region that the follicular stem cells in the bulge region

are intact and are not the only reservoir of stem are intact and are not the only reservoir of stem

cells in the hair follicle.cells in the hair follicle.

The preservation of CK15 expression in AGA The preservation of CK15 expression in AGA

supports the concept that it is a nonscarring supports the concept that it is a nonscarring

type of alopecia and suggests its potential type of alopecia and suggests its potential

reversibility.reversibility.

The loss of CD34+ cells in AGA provides insight The loss of CD34+ cells in AGA provides insight

into the possible mechanism of miniaturization into the possible mechanism of miniaturization

and supports the notion that a defect in and supports the notion that a defect in

conversion of bulge stem cells to progenitor conversion of bulge stem cells to progenitor

cells may play a role in disease progression. cells may play a role in disease progression.

Also the decreased expression of P63 in this Also the decreased expression of P63 in this

study suggests a role of this protein in the study suggests a role of this protein in the

pathogenesis of AGA.pathogenesis of AGA.

Future studies are recommended Future studies are recommended using other stem cell markers to using other stem cell markers to further clarify the role of follicular further clarify the role of follicular stem cells in androgenetic alopecia stem cells in androgenetic alopecia and to identify signals responsible and to identify signals responsible for transformation of these stem for transformation of these stem cells to progenitor cells.cells to progenitor cells.