# Preclinical Evidence for the Benefits of Penile Rehabilitation Therapy following Nerve-Sparing Radical Prostatectomy

Authors: M. Albersen; S. Joniau; H. Claes; H. Van Poppel
Journal: Advances in Urology
 (2008)

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## Abstract

Erectile dysfunction following radical prostatectomy remains a frequent problem despite the development of nerve-sparing techniques. This erectile dysfunction is believed to be neurogenic, enhanced by hypoxia-induced structural changes which result in additional veno-occlusive dysfunction. Recently, daily use of intracavernous vasoactive substances and oral use of PDE5-inhibitors have been clinically studied for treatment of postprostatectomy erectile dysfunction. Since these studies showed benefits of “penile rehabilitation therapy,” these effects have been studied in a preclinical setting. We reviewed experimental literature on erectile tissue preserving and neuroregenerative treatment strategies, and found that preservation of the erectile tissue by the use of intracavernous nitric oxide donors or vasoactive substances, oral PDE5-inhibitors, and hyperbaric oxygen therapy improved erectile function by antifibrotic effects and preservation of smooth muscle. Furthermore, neuroregenerative strategies using neuroimmunophilin ligands, neurotrophins, growth factors, and stem cell therapy show improved erectile function by preservation of NOS-containing nerve fibers.


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## Body

## 1. Introduction

Cancer of the prostate is now recognized as one of the
principal medical problems in the male population [1]. At present, radical prostatectomy
(RP), either retropubic, perineal, laparoscopic, or robot-assisted
laparoscopic, is the treatment of choice in young men with clinically localised
prostate cancer. Since prostate cancer is detected at increasingly younger age
and lower stage, patients undergoing radical prostatectomy generally have good
baseline erectile function, and have high expectations concerning the
preservation of erectile function following the procedure. Since Walsh and
Donker published their insights in the etiology and prevention of impotence
following retropubic RP in 1982, the nerve-sparing technique they described is
widely employed to improve postoperative erectile function [2, 3]. Literature research reveals widely disparate potency
rates between various studies concerning nerve-sparing RP (6–86%) [4–12]. Despite the development of new techniques for
preservation of the cavernous nerves, many men
continue to suffer from erectile dysfunction (ED) and penile shortening after
RP, due to neuropraxia. In 1997, Montorsi
and colleagues introduced the concept of early postoperative vasoactive therapy
and penile rehabilitation, and they suggested that the early postoperative use
of intracavernosal injection therapy to promote penile erection may result in
improved erectile function outcomes [13].  More recently, PDE-5-inhibitors have
been studied for their use in penile rehabilitation [14, 15]. Good results have
been achieved with the use of nerve grafts in nonnerve sparing surgery, and the
use of neuroregenerative tubular implants [16, 17]. This review article attempts
to summarize the contemporary basic scientific knowledge on the
pathophysiological mechanisms of post-RP ED and to review current basic science
evidence for medicinal, nonchirurgical penile rehabilitation therapy and
neuroregenerative therapies.


## 2. Anatomy and Physiology of the Erection

The penile corpora cavernosa and the corpus spongiosum
are innervated by the combined sympathetic and parasympathetic cavernous nerves
(CNs), which arise from the pelvic plexus. These nerves are condensed in the
neurovascular bundles (NVBs), which can be found in close relation to the
dorsolateral side of the prostatic capsula and urethra, although recent
findings suggest that a significant number of nerves can be found along the
ventral parts of the prostatic capsula (i.e., ‘‘outside’’ the classic NVB)
[18–20]. Nitric oxide (NO), released during nonadrenergic, noncholinergic
(NANC) stimulation from the CN terminals and from the endothelium is
the principal neurotransmitter mediating penile erection. NO
activates guanylyl cyclase, an enzyme that raises the intracellular concentration
of cyclic guanosine monophosphate (cGMP), leading to activation of cGMP
specific protein kinases which activate further intracellular events,
eventually leading to reduction of intracellular calcium, and relaxation of the
smooth muscle. cGMP is hydrolysedto GMP by phosphodiesterase type 5
during return to the flaccid state. The vasodilator prostaglandin E1 (PGE1)
also causes SM relaxation but by increasing the concentration of the cyclic
adenosine monophosphate (cAMP), via stimulation of adenylate cyclase. The
resulting vasodilation results in an increase in penile blood flow. During this
phase of tumescence, relaxation of the trabecular smooth muscle increases the
compliance of the sinusoids, resulting in expansion of the
sinusoidal system. The subtunical venous plexusesare thus
compressed between the expanding sinusoidal wall and the noncompliant tunica
albuginea, resulting in almost total subtunical venous occlusion.
These events, augmented by contraction of the ischiocavernosus
muscles, trap the blood within the corpora cavernosa, with an
intracavernous pressure that can approach several hundreds of mm Hg [21–24].


## 3. Pathophysiologic Mechanisms of Erectile Dysfunction Following Radical Prostatectomy

### 3.1. Impairment in Corpus Cavernosum Oxygenation

#### 3.1.1. Physiological Changes in Penile Oxygen Tension

Azadzoi and colleagues showed in a
canine model that subtunical oxygen tension in theflaccid penis was close to 100 mmHg, consistent with a largely
arterial circulation, whereas deep cavernosal oxygen tension measurements
showed an oxygen tension consistent with venous blood flow. With pelvic nerve
stimulation or injection of vasoactive agents, oxygen tension deep within the
corpus cavernosum increased during penile erection from a level consistent with venous blood to a level consistent
with arterial blood. Also, due to the increase of intracavernosal pressure,
blood flow in the subtunical blood flow significantly decreased [25]. Various
investigators state that a persistent flaccid state of the penis, as seen after
cavernous neurotomy with absence of nocturnal erections, causes a relatively
hypoxic state causing a variety of structural and functional changes in the
corpora cavernosa.


#### 3.1.2. Nocturnal Penile Tumescence (NPT)

Nocturnal penile tumescence is a sleep-related
phenomenon that occurs during the rapid eye movement sleep-phase in men without
erectile dysfunction. These nocturnal erections occur 3–5 times per night,
lasting 30–45 minutes [26]. During these phases of NPT, arterial oxygen is
supplied to the cavernous tissue. This supply of oxygen is necessary for the
formation of NO by nitric oxide synthase (NOS) in the cavernosal endothelium
(eNOS) and in the CN endings (nNOS). Upon its release, NO diffuses locally into
smooth muscle cells and binds with its physiologic receptor, soluble guanylyl
cyclase, which catalyses the conversion of GTP to cGMP. This second messenger
eventually evokes a decline in cytosolic calcium resulting in smooth muscle
relaxation and penile tumescence [20, 24]. Bannowsky et al. showed absence of
NPT in a control group without nerve-sparing versus 97% NPT in the group that
underwent nerve sparing RP [27]. This absence of NPT following cavernous
neurotomy causes a permanent lack of free oxygen in the corpus cavernosum,
resulting in a predisposition to cavernosal fibrosis [19, 28]. In a rat model,
this neurotomy-induced hypoxia was demonstated by significantly higher protein
expression and immunohistochemical staining of hypoxia-inducible factor-1α
(HIF-1α) in the neurotomised rat [28]. HIF-1α is a transcription factor that is
expressed when mammalian cells are subjected to hypoxia. This cytokine
activates the transcription of genes encoding proteins that are important for
maintaining oxygen homeostasis. This finding confirms the theory that hypoxia
of rat penes after iatrogenic nerve injury was induced by the loss of nocturnal
erections [29].


#### 3.1.3.  Altered Contractility of Penile Smooth Muscle by Penile Oxygenation

Various investigators have shown
that responses of blood vessels to NO were inhibited at low oxygen tensions
[30–34]. Kim et al. have studied this effect of oxygen tension on the
resistance of the arterial bed of the penis and on the relaxation of cavernosal
trabecular smooth muscle. They showed that isolated human and rabbit corpus
cavernosum tissue strips relaxed after addition of endothelium-dependent
dilator acetylcholine and to electrical stimulation of the autonomic nerves,
when bathed in an organ-bath with arterial-like PO2. These (NO-mediated) responses
progressively were inhibited when PO2 levels where decreased to levels measured in the
deep corpus cavernosum of the flaccid penis. Restoring PO2 levels to normoxic
conditions restored this endothelium-dependent and neurogenic relaxation.
Furthermore, they showed that low PO2 reduced basal levels of cGMP, prevented cGMP
accumulation induced by nerve stimulation, and inhibited cytosolic NOS
activity. They concluded that penile concentration of oxygen modulates penile
erection by regulating NO synthesis in the corpus cavernosum [35].


### 3.2. Structural Changes Following Cavernous Neurotomy

A number of patients who have undergone radical
prostatectomy complain of subjective penile shortening. Various clinical
studies have addressed this finding, with a reported incidence of penile
shortening of in 9–71% of patients [36–39]. User et al. showed a loss in penile
wet weight in a rat model following bilateral neurotomy, although unilateral
neurotomy showed a much greater preservation of penile weight. The majority of
authors suggest that penile fibrosis accounts for these morphometric changes,
by reducing the extensibility and volume of the corpus cavernosum [40]. Also,
denervation-induced apoptosis has been proposed to play a role in penile
shortening after neurotomy [41]. Mulhall recently proposed a hypothesis that
penile size reduction should be divided into early postoperative and reversible
size reduction (hypothised to be due to postoperative sympathetic overdrive),
and delayed and permanent structural changes due to hypoxia and
denervation-induced structural changes [42].


#### 3.2.1. Postoperative Early Reversible Penile Size Reduction

The hypothesis of Mulhall that early penile size
reduction is the result of sympathetic hyperinnervation is based on the fact
that nerve growth factor (NGF), a neurotrophin which is released after
peripheral nerve damage, induces differentiation of sympathetic neurons and
enhances target innervation [43, 44]. This mechanism is suspected to induce
sympathetic hyperinnervation of the corpus cavernosum, following an earlier
regeneration of sympathetic nerve fibers, resulting in smooth muscle
contraction and a hypertonic state of the penis. This hypothesis was supported
by a recently published clinical study of Gontero and colleagues [39].


#### 3.2.2. Postoperative Permanent Structural Changes: Apoptosis

Klein et al. stated that, given the observation that
penile size may decrease after radical prostatectomy, damage to the cavernous
nerves might result in apoptosis of cells in erectile tissues of the penis.
They found the presence of condensed and fragmented cell nuclei (characteristic
of apoptotic cells) within the erectile tissue, and expression of sulfated
glycoprotein-2 (a protein found to increase dramatically in several urogenital
tract tissues undergoing apoptosis), in denervated rat-penises versus a sham-operated
control group [41]. User and colleagues have elucidated the role of apoptosis
in the pathophysiology of postprostatectomy ED in a postpubertal male
Sprague-Dawley rat model. Rats were randomized to bilateral or unilateral CN
transection versus a sham operation. At different time intervals following the
procedure, penile wet weight, DNA content, and protein content were measured.
Tissue sections of the penis were stained for apoptosis, and the apoptotic
index was calculated. Finally, staining for endothelial and smooth muscle cells
was done to identify the apoptotic cell line. They found that bilateral
cavernous neurotomy induced significant apoptosis. In addition, it was found that
most apoptotic cells were located just beneath the tunica albuginea where the
subtunical venular plexus is located. Finally the authors demonstrated that
apoptotic cells were predominantly smooth muscle cells and not endothelial
cells [40, 45]. This finding strongly implies that smooth muscle cells somehow
depend on neural innervation. The programmed cell-death of smooth muscle has further
implications. It has been shown that reductions in the quantity of smooth
muscle cells is a pivotal event in the development of veno-occlusive
dysfunction, which has been shown to play a major role in postprostatectomy ED
[46, 47].Sonic hedgehog homolog (SHH) is one of three proteins in the mammalian
hedgehog family. It plays a key role in regulating vertebrate organogenesis and
remains important in the adult. It controls cell division of adult stem cells.
SHH was suggested to play a role in apoptosis of smooth muscle cells. In a
series of experiments by Podlascek et al., bilateral CN resection resulted in
decreased SHH in the smooth muscle of the corpora cavernosal sinusoids and
extensive morphological changes, including increased apoptosis. The SHH cascade acts to establish normal
penile morphology. Nerve injury disrupts the SHH cascade and corpora cavernosal homeostasis such that
morphological changes in sinusoid structure ensue, resulting in ED. Bond et al.
illustrated that neural activity and a trophic factor from the pelvic ganglia
or cavernous nerves are necessary to regulate SHH protein and smooth muscle abundance
in the penis [48–51].


#### 3.2.3. Postoperative Permanent Structural Changes: Fibrosis

Leungwattanakij and associates evaluated the isolated
effects of CN injury on cavernous oxygenation and cavernous fibrosis using the
Sprague-Dawley rat. They showed that production of transforming growth factor-β1 (TGF-β1) and TGF-β1 mRNA expression as well as collagen I
and III protein expressions was
significantly greater in the cavernous neurotomy-group compared to a
sham-operated group [28]. TGF-β1 is associated with increased
collagen synthesis by cavernous smooth muscle cells, resulting in cavernosal
fibrosis. Furthermore, TGF-β1 dependent endothelin-1 (ET-1)
synthesis is increased. ET-1 is a potent constrictor of penile smooth muscle
and a profibrotic peptide [52]. Studies also showed that low oxygen tension in
human cavernosal tissue inhibits production of PGE1. PGE1 inhibits collagen
formation by inhibiting TGF-β1 that induces collagen synthesis.
With the inhibition of PGE1, TGF-β1 is allowed to induce connective tissue
synthesis [53–59]. Prolonged periods of cavernosal hypoxia thus lead to
increased deposition of connective tissue that may finally lead to a decrease
in penile distensibility, veno-occlusive dysfunction, ED, and reduction in
penile length.


## 3.1. Impairment in Corpus Cavernosum Oxygenation

### 3.1.1. Physiological Changes in Penile Oxygen Tension

Azadzoi and colleagues showed in a
canine model that subtunical oxygen tension in theflaccid penis was close to 100 mmHg, consistent with a largely
arterial circulation, whereas deep cavernosal oxygen tension measurements
showed an oxygen tension consistent with venous blood flow. With pelvic nerve
stimulation or injection of vasoactive agents, oxygen tension deep within the
corpus cavernosum increased during penile erection from a level consistent with venous blood to a level consistent
with arterial blood. Also, due to the increase of intracavernosal pressure,
blood flow in the subtunical blood flow significantly decreased [25]. Various
investigators state that a persistent flaccid state of the penis, as seen after
cavernous neurotomy with absence of nocturnal erections, causes a relatively
hypoxic state causing a variety of structural and functional changes in the
corpora cavernosa.


### 3.1.2. Nocturnal Penile Tumescence (NPT)

Nocturnal penile tumescence is a sleep-related
phenomenon that occurs during the rapid eye movement sleep-phase in men without
erectile dysfunction. These nocturnal erections occur 3–5 times per night,
lasting 30–45 minutes [26]. During these phases of NPT, arterial oxygen is
supplied to the cavernous tissue. This supply of oxygen is necessary for the
formation of NO by nitric oxide synthase (NOS) in the cavernosal endothelium
(eNOS) and in the CN endings (nNOS). Upon its release, NO diffuses locally into
smooth muscle cells and binds with its physiologic receptor, soluble guanylyl
cyclase, which catalyses the conversion of GTP to cGMP. This second messenger
eventually evokes a decline in cytosolic calcium resulting in smooth muscle
relaxation and penile tumescence [20, 24]. Bannowsky et al. showed absence of
NPT in a control group without nerve-sparing versus 97% NPT in the group that
underwent nerve sparing RP [27]. This absence of NPT following cavernous
neurotomy causes a permanent lack of free oxygen in the corpus cavernosum,
resulting in a predisposition to cavernosal fibrosis [19, 28]. In a rat model,
this neurotomy-induced hypoxia was demonstated by significantly higher protein
expression and immunohistochemical staining of hypoxia-inducible factor-1α
(HIF-1α) in the neurotomised rat [28]. HIF-1α is a transcription factor that is
expressed when mammalian cells are subjected to hypoxia. This cytokine
activates the transcription of genes encoding proteins that are important for
maintaining oxygen homeostasis. This finding confirms the theory that hypoxia
of rat penes after iatrogenic nerve injury was induced by the loss of nocturnal
erections [29].


### 3.1.3.  Altered Contractility of Penile Smooth Muscle by Penile Oxygenation

Various investigators have shown
that responses of blood vessels to NO were inhibited at low oxygen tensions
[30–34]. Kim et al. have studied this effect of oxygen tension on the
resistance of the arterial bed of the penis and on the relaxation of cavernosal
trabecular smooth muscle. They showed that isolated human and rabbit corpus
cavernosum tissue strips relaxed after addition of endothelium-dependent
dilator acetylcholine and to electrical stimulation of the autonomic nerves,
when bathed in an organ-bath with arterial-like PO2. These (NO-mediated) responses
progressively were inhibited when PO2 levels where decreased to levels measured in the
deep corpus cavernosum of the flaccid penis. Restoring PO2 levels to normoxic
conditions restored this endothelium-dependent and neurogenic relaxation.
Furthermore, they showed that low PO2 reduced basal levels of cGMP, prevented cGMP
accumulation induced by nerve stimulation, and inhibited cytosolic NOS
activity. They concluded that penile concentration of oxygen modulates penile
erection by regulating NO synthesis in the corpus cavernosum [35].


## 3.1.1. Physiological Changes in Penile Oxygen Tension

Azadzoi and colleagues showed in a
canine model that subtunical oxygen tension in theflaccid penis was close to 100 mmHg, consistent with a largely
arterial circulation, whereas deep cavernosal oxygen tension measurements
showed an oxygen tension consistent with venous blood flow. With pelvic nerve
stimulation or injection of vasoactive agents, oxygen tension deep within the
corpus cavernosum increased during penile erection from a level consistent with venous blood to a level consistent
with arterial blood. Also, due to the increase of intracavernosal pressure,
blood flow in the subtunical blood flow significantly decreased [25]. Various
investigators state that a persistent flaccid state of the penis, as seen after
cavernous neurotomy with absence of nocturnal erections, causes a relatively
hypoxic state causing a variety of structural and functional changes in the
corpora cavernosa.


## 3.1.2. Nocturnal Penile Tumescence (NPT)

Nocturnal penile tumescence is a sleep-related
phenomenon that occurs during the rapid eye movement sleep-phase in men without
erectile dysfunction. These nocturnal erections occur 3–5 times per night,
lasting 30–45 minutes [26]. During these phases of NPT, arterial oxygen is
supplied to the cavernous tissue. This supply of oxygen is necessary for the
formation of NO by nitric oxide synthase (NOS) in the cavernosal endothelium
(eNOS) and in the CN endings (nNOS). Upon its release, NO diffuses locally into
smooth muscle cells and binds with its physiologic receptor, soluble guanylyl
cyclase, which catalyses the conversion of GTP to cGMP. This second messenger
eventually evokes a decline in cytosolic calcium resulting in smooth muscle
relaxation and penile tumescence [20, 24]. Bannowsky et al. showed absence of
NPT in a control group without nerve-sparing versus 97% NPT in the group that
underwent nerve sparing RP [27]. This absence of NPT following cavernous
neurotomy causes a permanent lack of free oxygen in the corpus cavernosum,
resulting in a predisposition to cavernosal fibrosis [19, 28]. In a rat model,
this neurotomy-induced hypoxia was demonstated by significantly higher protein
expression and immunohistochemical staining of hypoxia-inducible factor-1α
(HIF-1α) in the neurotomised rat [28]. HIF-1α is a transcription factor that is
expressed when mammalian cells are subjected to hypoxia. This cytokine
activates the transcription of genes encoding proteins that are important for
maintaining oxygen homeostasis. This finding confirms the theory that hypoxia
of rat penes after iatrogenic nerve injury was induced by the loss of nocturnal
erections [29].


## 3.1.3.  Altered Contractility of Penile Smooth Muscle by Penile Oxygenation

Various investigators have shown
that responses of blood vessels to NO were inhibited at low oxygen tensions
[30–34]. Kim et al. have studied this effect of oxygen tension on the
resistance of the arterial bed of the penis and on the relaxation of cavernosal
trabecular smooth muscle. They showed that isolated human and rabbit corpus
cavernosum tissue strips relaxed after addition of endothelium-dependent
dilator acetylcholine and to electrical stimulation of the autonomic nerves,
when bathed in an organ-bath with arterial-like PO2. These (NO-mediated) responses
progressively were inhibited when PO2 levels where decreased to levels measured in the
deep corpus cavernosum of the flaccid penis. Restoring PO2 levels to normoxic
conditions restored this endothelium-dependent and neurogenic relaxation.
Furthermore, they showed that low PO2 reduced basal levels of cGMP, prevented cGMP
accumulation induced by nerve stimulation, and inhibited cytosolic NOS
activity. They concluded that penile concentration of oxygen modulates penile
erection by regulating NO synthesis in the corpus cavernosum [35].


## 3.2. Structural Changes Following Cavernous Neurotomy

A number of patients who have undergone radical
prostatectomy complain of subjective penile shortening. Various clinical
studies have addressed this finding, with a reported incidence of penile
shortening of in 9–71% of patients [36–39]. User et al. showed a loss in penile
wet weight in a rat model following bilateral neurotomy, although unilateral
neurotomy showed a much greater preservation of penile weight. The majority of
authors suggest that penile fibrosis accounts for these morphometric changes,
by reducing the extensibility and volume of the corpus cavernosum [40]. Also,
denervation-induced apoptosis has been proposed to play a role in penile
shortening after neurotomy [41]. Mulhall recently proposed a hypothesis that
penile size reduction should be divided into early postoperative and reversible
size reduction (hypothised to be due to postoperative sympathetic overdrive),
and delayed and permanent structural changes due to hypoxia and
denervation-induced structural changes [42].


### 3.2.1. Postoperative Early Reversible Penile Size Reduction

The hypothesis of Mulhall that early penile size
reduction is the result of sympathetic hyperinnervation is based on the fact
that nerve growth factor (NGF), a neurotrophin which is released after
peripheral nerve damage, induces differentiation of sympathetic neurons and
enhances target innervation [43, 44]. This mechanism is suspected to induce
sympathetic hyperinnervation of the corpus cavernosum, following an earlier
regeneration of sympathetic nerve fibers, resulting in smooth muscle
contraction and a hypertonic state of the penis. This hypothesis was supported
by a recently published clinical study of Gontero and colleagues [39].


### 3.2.2. Postoperative Permanent Structural Changes: Apoptosis

Klein et al. stated that, given the observation that
penile size may decrease after radical prostatectomy, damage to the cavernous
nerves might result in apoptosis of cells in erectile tissues of the penis.
They found the presence of condensed and fragmented cell nuclei (characteristic
of apoptotic cells) within the erectile tissue, and expression of sulfated
glycoprotein-2 (a protein found to increase dramatically in several urogenital
tract tissues undergoing apoptosis), in denervated rat-penises versus a sham-operated
control group [41]. User and colleagues have elucidated the role of apoptosis
in the pathophysiology of postprostatectomy ED in a postpubertal male
Sprague-Dawley rat model. Rats were randomized to bilateral or unilateral CN
transection versus a sham operation. At different time intervals following the
procedure, penile wet weight, DNA content, and protein content were measured.
Tissue sections of the penis were stained for apoptosis, and the apoptotic
index was calculated. Finally, staining for endothelial and smooth muscle cells
was done to identify the apoptotic cell line. They found that bilateral
cavernous neurotomy induced significant apoptosis. In addition, it was found that
most apoptotic cells were located just beneath the tunica albuginea where the
subtunical venular plexus is located. Finally the authors demonstrated that
apoptotic cells were predominantly smooth muscle cells and not endothelial
cells [40, 45]. This finding strongly implies that smooth muscle cells somehow
depend on neural innervation. The programmed cell-death of smooth muscle has further
implications. It has been shown that reductions in the quantity of smooth
muscle cells is a pivotal event in the development of veno-occlusive
dysfunction, which has been shown to play a major role in postprostatectomy ED
[46, 47].Sonic hedgehog homolog (SHH) is one of three proteins in the mammalian
hedgehog family. It plays a key role in regulating vertebrate organogenesis and
remains important in the adult. It controls cell division of adult stem cells.
SHH was suggested to play a role in apoptosis of smooth muscle cells. In a
series of experiments by Podlascek et al., bilateral CN resection resulted in
decreased SHH in the smooth muscle of the corpora cavernosal sinusoids and
extensive morphological changes, including increased apoptosis. The SHH cascade acts to establish normal
penile morphology. Nerve injury disrupts the SHH cascade and corpora cavernosal homeostasis such that
morphological changes in sinusoid structure ensue, resulting in ED. Bond et al.
illustrated that neural activity and a trophic factor from the pelvic ganglia
or cavernous nerves are necessary to regulate SHH protein and smooth muscle abundance
in the penis [48–51].


### 3.2.3. Postoperative Permanent Structural Changes: Fibrosis

Leungwattanakij and associates evaluated the isolated
effects of CN injury on cavernous oxygenation and cavernous fibrosis using the
Sprague-Dawley rat. They showed that production of transforming growth factor-β1 (TGF-β1) and TGF-β1 mRNA expression as well as collagen I
and III protein expressions was
significantly greater in the cavernous neurotomy-group compared to a
sham-operated group [28]. TGF-β1 is associated with increased
collagen synthesis by cavernous smooth muscle cells, resulting in cavernosal
fibrosis. Furthermore, TGF-β1 dependent endothelin-1 (ET-1)
synthesis is increased. ET-1 is a potent constrictor of penile smooth muscle
and a profibrotic peptide [52]. Studies also showed that low oxygen tension in
human cavernosal tissue inhibits production of PGE1. PGE1 inhibits collagen
formation by inhibiting TGF-β1 that induces collagen synthesis.
With the inhibition of PGE1, TGF-β1 is allowed to induce connective tissue
synthesis [53–59]. Prolonged periods of cavernosal hypoxia thus lead to
increased deposition of connective tissue that may finally lead to a decrease
in penile distensibility, veno-occlusive dysfunction, ED, and reduction in
penile length.


## 3.2.1. Postoperative Early Reversible Penile Size Reduction

The hypothesis of Mulhall that early penile size
reduction is the result of sympathetic hyperinnervation is based on the fact
that nerve growth factor (NGF), a neurotrophin which is released after
peripheral nerve damage, induces differentiation of sympathetic neurons and
enhances target innervation [43, 44]. This mechanism is suspected to induce
sympathetic hyperinnervation of the corpus cavernosum, following an earlier
regeneration of sympathetic nerve fibers, resulting in smooth muscle
contraction and a hypertonic state of the penis. This hypothesis was supported
by a recently published clinical study of Gontero and colleagues [39].


## 3.2.2. Postoperative Permanent Structural Changes: Apoptosis

Klein et al. stated that, given the observation that
penile size may decrease after radical prostatectomy, damage to the cavernous
nerves might result in apoptosis of cells in erectile tissues of the penis.
They found the presence of condensed and fragmented cell nuclei (characteristic
of apoptotic cells) within the erectile tissue, and expression of sulfated
glycoprotein-2 (a protein found to increase dramatically in several urogenital
tract tissues undergoing apoptosis), in denervated rat-penises versus a sham-operated
control group [41]. User and colleagues have elucidated the role of apoptosis
in the pathophysiology of postprostatectomy ED in a postpubertal male
Sprague-Dawley rat model. Rats were randomized to bilateral or unilateral CN
transection versus a sham operation. At different time intervals following the
procedure, penile wet weight, DNA content, and protein content were measured.
Tissue sections of the penis were stained for apoptosis, and the apoptotic
index was calculated. Finally, staining for endothelial and smooth muscle cells
was done to identify the apoptotic cell line. They found that bilateral
cavernous neurotomy induced significant apoptosis. In addition, it was found that
most apoptotic cells were located just beneath the tunica albuginea where the
subtunical venular plexus is located. Finally the authors demonstrated that
apoptotic cells were predominantly smooth muscle cells and not endothelial
cells [40, 45]. This finding strongly implies that smooth muscle cells somehow
depend on neural innervation. The programmed cell-death of smooth muscle has further
implications. It has been shown that reductions in the quantity of smooth
muscle cells is a pivotal event in the development of veno-occlusive
dysfunction, which has been shown to play a major role in postprostatectomy ED
[46, 47].Sonic hedgehog homolog (SHH) is one of three proteins in the mammalian
hedgehog family. It plays a key role in regulating vertebrate organogenesis and
remains important in the adult. It controls cell division of adult stem cells.
SHH was suggested to play a role in apoptosis of smooth muscle cells. In a
series of experiments by Podlascek et al., bilateral CN resection resulted in
decreased SHH in the smooth muscle of the corpora cavernosal sinusoids and
extensive morphological changes, including increased apoptosis. The SHH cascade acts to establish normal
penile morphology. Nerve injury disrupts the SHH cascade and corpora cavernosal homeostasis such that
morphological changes in sinusoid structure ensue, resulting in ED. Bond et al.
illustrated that neural activity and a trophic factor from the pelvic ganglia
or cavernous nerves are necessary to regulate SHH protein and smooth muscle abundance
in the penis [48–51].


## 3.2.3. Postoperative Permanent Structural Changes: Fibrosis

Leungwattanakij and associates evaluated the isolated
effects of CN injury on cavernous oxygenation and cavernous fibrosis using the
Sprague-Dawley rat. They showed that production of transforming growth factor-β1 (TGF-β1) and TGF-β1 mRNA expression as well as collagen I
and III protein expressions was
significantly greater in the cavernous neurotomy-group compared to a
sham-operated group [28]. TGF-β1 is associated with increased
collagen synthesis by cavernous smooth muscle cells, resulting in cavernosal
fibrosis. Furthermore, TGF-β1 dependent endothelin-1 (ET-1)
synthesis is increased. ET-1 is a potent constrictor of penile smooth muscle
and a profibrotic peptide [52]. Studies also showed that low oxygen tension in
human cavernosal tissue inhibits production of PGE1. PGE1 inhibits collagen
formation by inhibiting TGF-β1 that induces collagen synthesis.
With the inhibition of PGE1, TGF-β1 is allowed to induce connective tissue
synthesis [53–59]. Prolonged periods of cavernosal hypoxia thus lead to
increased deposition of connective tissue that may finally lead to a decrease
in penile distensibility, veno-occlusive dysfunction, ED, and reduction in
penile length.


## 4. Corpus Cavernosum Oxygenation Preserving Strategies

### 4.1. Intracavernous Injections of Vasoactive Agents

The finding that poor cavernosal
oxygenation induces cavernosal structural damage which causes erectile
dysfunction following radical prostatectomy leads to the hypothesis that
preserving the oxygenation of the corpora cavernosa by improving blood flow
might improve erectile dysfunction. Montorsi et al. conducted a pioneering
clinical study on the effects of intracavernosal injections of aldoprostil
early after bilateral nerve sparing prostatectomy and saw a significantly
higher recovery of spontaneous erections in treated patients treated versus
observation alone [13].Preclinical data on the use of intracavernosal and intraurethral
vasoactive agents is limited. Moreland et al. found that in vitro TGF-β1 mRNA
induction was suppressed by PGE1 via increased levels of cAMP. These data
suggest that prostaglandins and TGF-β1 may play a key role in
modulation of collagen synthesis in the corpus cavernosum, and in the
regulation of fibrosis of the corpus cavernosum [55–60]. A cross-regulation of
intracellular cGMP and cAMP in cultured human corpus cavernosum smooth muscle
cells was found by Kim et al. They showed a rise in cGMP (which has been
identified as an antifibrotic mediator) as a response to a rise in
intracellular cAMP, which is accomplished by intracavernous injection of PGE1
[61, 62]. These in vitro observations are likely to play a major role in the
recovery of spontaneous erections in patients treated with intracavernous PGE1
following nerve sparing radical prostatectomy.


### 4.2. PDE-5-Inhibitors (PDE5-I)

PDE5-I act within the smooth muscle
cells where the enzyme phosphodiesterase-5, an enzyme which naturally degrades
cGMP, is inhibited. This causes increased levels of cGMP, leading to activation
of cGMP specific protein kinases which activate further intracellular events,
eventually leading to reduction of intracellular calcium, being associated with
cavernosal smooth muscle relaxation. The mechanism of PDE5-I is thus dependent
on the production of NO from the nerve endings, which initiates production of
cGMP.Padma-Nathan and colleagues reported the results of a
randomized, placebo-controlled study examining the benefits of nightly
administration of sildenafil during the postoperative period following a
bilateral nerve-sparing RRP. This study included 76 men with normal
preoperative erectile function. Forty-eight weeks after bilateral nerve-sparing
RRP, 27% of patients receiving sildenafil demonstrated return of spontaneous
erectile function, compared with 4% in the placebo group. Postoperative NPT
assessments supported these patient-reported results [15]. Since Padma-Nathan
et al. published their results of the clinical applicability of PDE5-I,
preclinical effects of this class of drugs have been the focus of intense
investigation.Ferrini et al. were the first to demonstrate that
long-term continuous oral administration of a PDE5-I (vardenafil) in rats
prevents neuropraxia-induced veno-occlusive dysfunction, presumably as a result
of the antifibrotic effects of PDE5-I and the preservation of the corporal SM
mass by upregulation of SM cell replication [63]. The same group demonstrated
the same antifibrotic effects and prevention of veno-occlusive dysfunction with
the use of the other PDE5-inhibitors, sildenafil and tadalafil, and showed that
these effects where cGMP mediated, but independent of inducible NOS (iNOS)
upregulation, as they hypothesised in their vardenafil experiment [62–64]. Vignozzi
et al. demonstrated in vitro that bilateral CN neurotomy induced penile hypoxia shown by a dramatically increased
hypoxyprobe labeling, a generally accepted probe of hypo-oxygenation after a
3-month interval from surgical neurotomy. They
found that chronic tadalafil treatment almost completely restored penile oxygenation and smooth muscle/fibrous tissue
ratio at histology [65]. They also found that sildanefil administration was
able to counteract penile hypoxia, expression of profibrotic endothelin-1 type
B receptor (ETB), and hypersensitivity to a ETB agonist IRL-1620 (using an in
vitro contractility study), with its effect being more evident the earlier it
was administered [66]. These studies indicate that PDE5-I can potentially
protect and preserve the integrity of the corpora after cavernosal nerve damage
when given on a long term and continuous basis [67].At genomic level, the impact of sildanefil treatment
on gene expression in the rat CN injury model was evaluated by Müller et al. of the Memorial Sloan-Kettering Cancer Centre in New York. They used microarray analysis to
elucidate which genes are altered by administration of PDE5-I in the CN injury
model and  identified genes that are
altered in the CN injury model and that are restored towards normal levels with
the use of sildenafil. These genes predominantly belonged to apoptosis,
neurotrophism, and endothelial funtion families [68].


### 4.3. Hyperbaric Oxygen Therapy

Hyperbaric oxygen therapy (HBOT) has been used in the
treatment of a variety of medical conditions, in the urology field especially
for radiation-induced cystitis. Oxygen hypersaturation has been proven to
induce angiogenesis and enhance wound healing based on the excessive supply of
oxygen. Since hypoxia is assumed to be the capital factor in inducing penile
structural changes and erectile dysfunction following cavernous nerve injury,
Müller et al. [69] investigated the effects of hyperbaric oxygen therapy,
inducing hyperoxygenation in this setting. They used a bilateral cavernous
nerve crush-injury Sprague-Dawley rat model which was exposed to ten sessions
of hyperbaric oxygen at three atmospheres for the duration 90 minutes. They measured ICP after
CN stimulation under controlled conditions with continuous systemic blood
pressure measurement, which according to the authors provides an accurate
measurement of erectile response. They found that rats in the HBOT group had
significantly higher mean ICP/MAP ratios compared with the untreated group.
They demonstrated preservation of smooth muscle/collagen ratio with HBOT using Masson’s Trichrome coloring of penile
cross-sections. This study was the first to show a protective effect of HBOT on
erectile function, and literature data is therefore limited [69]. Theoretically, induction of angiogenesis
by HBOT might potentially accelerate tumor growth in cancer patients and
could therefore be unfavourable in patients treated for pelvic malignancies.
This issue was however studied in a mouse model that was developed to
investigate the effect of HBOT on prostate cancer cell growth in patients
treated for radiation-induced hemorrhagic cystitis. The investigators did not
witness increased rates of tumor growth, and they concluded that HBOT in cases
of prostate cancer is not associated with increased oncological risk [70].


### 4.4. Nitric Oxide Donor Therapy

Since neuropraxia causes loss of NOS
containing nerves and hypoxia, resulting in a decrease in available NO and
structural changes, Veliev and associates hypothesised that direct restoration
of NO in the penis could prevent these changes after denervation. This group
investigated the effect of intracorporeal administration of new NO donor
dinitrosyl iron complex in a bilateral CN crush injury group compared to a
sham-operated group and an untreated group CN crush injury group. They used analysis
of mitotic activity of smooth muscle (decreased after CN injury), endothelium,
and fibroblasts (both increased after CN injury) for evaluation of cavernous
fibrosis development and found a stabilisation of fibroblast proliferation and
improvement of smooth muscle mitotic activity in the group treated with
intracavernosal injections of NO donor [71].


## 4.1. Intracavernous Injections of Vasoactive Agents

The finding that poor cavernosal
oxygenation induces cavernosal structural damage which causes erectile
dysfunction following radical prostatectomy leads to the hypothesis that
preserving the oxygenation of the corpora cavernosa by improving blood flow
might improve erectile dysfunction. Montorsi et al. conducted a pioneering
clinical study on the effects of intracavernosal injections of aldoprostil
early after bilateral nerve sparing prostatectomy and saw a significantly
higher recovery of spontaneous erections in treated patients treated versus
observation alone [13].Preclinical data on the use of intracavernosal and intraurethral
vasoactive agents is limited. Moreland et al. found that in vitro TGF-β1 mRNA
induction was suppressed by PGE1 via increased levels of cAMP. These data
suggest that prostaglandins and TGF-β1 may play a key role in
modulation of collagen synthesis in the corpus cavernosum, and in the
regulation of fibrosis of the corpus cavernosum [55–60]. A cross-regulation of
intracellular cGMP and cAMP in cultured human corpus cavernosum smooth muscle
cells was found by Kim et al. They showed a rise in cGMP (which has been
identified as an antifibrotic mediator) as a response to a rise in
intracellular cAMP, which is accomplished by intracavernous injection of PGE1
[61, 62]. These in vitro observations are likely to play a major role in the
recovery of spontaneous erections in patients treated with intracavernous PGE1
following nerve sparing radical prostatectomy.


## 4.2. PDE-5-Inhibitors (PDE5-I)

PDE5-I act within the smooth muscle
cells where the enzyme phosphodiesterase-5, an enzyme which naturally degrades
cGMP, is inhibited. This causes increased levels of cGMP, leading to activation
of cGMP specific protein kinases which activate further intracellular events,
eventually leading to reduction of intracellular calcium, being associated with
cavernosal smooth muscle relaxation. The mechanism of PDE5-I is thus dependent
on the production of NO from the nerve endings, which initiates production of
cGMP.Padma-Nathan and colleagues reported the results of a
randomized, placebo-controlled study examining the benefits of nightly
administration of sildenafil during the postoperative period following a
bilateral nerve-sparing RRP. This study included 76 men with normal
preoperative erectile function. Forty-eight weeks after bilateral nerve-sparing
RRP, 27% of patients receiving sildenafil demonstrated return of spontaneous
erectile function, compared with 4% in the placebo group. Postoperative NPT
assessments supported these patient-reported results [15]. Since Padma-Nathan
et al. published their results of the clinical applicability of PDE5-I,
preclinical effects of this class of drugs have been the focus of intense
investigation.Ferrini et al. were the first to demonstrate that
long-term continuous oral administration of a PDE5-I (vardenafil) in rats
prevents neuropraxia-induced veno-occlusive dysfunction, presumably as a result
of the antifibrotic effects of PDE5-I and the preservation of the corporal SM
mass by upregulation of SM cell replication [63]. The same group demonstrated
the same antifibrotic effects and prevention of veno-occlusive dysfunction with
the use of the other PDE5-inhibitors, sildenafil and tadalafil, and showed that
these effects where cGMP mediated, but independent of inducible NOS (iNOS)
upregulation, as they hypothesised in their vardenafil experiment [62–64]. Vignozzi
et al. demonstrated in vitro that bilateral CN neurotomy induced penile hypoxia shown by a dramatically increased
hypoxyprobe labeling, a generally accepted probe of hypo-oxygenation after a
3-month interval from surgical neurotomy. They
found that chronic tadalafil treatment almost completely restored penile oxygenation and smooth muscle/fibrous tissue
ratio at histology [65]. They also found that sildanefil administration was
able to counteract penile hypoxia, expression of profibrotic endothelin-1 type
B receptor (ETB), and hypersensitivity to a ETB agonist IRL-1620 (using an in
vitro contractility study), with its effect being more evident the earlier it
was administered [66]. These studies indicate that PDE5-I can potentially
protect and preserve the integrity of the corpora after cavernosal nerve damage
when given on a long term and continuous basis [67].At genomic level, the impact of sildanefil treatment
on gene expression in the rat CN injury model was evaluated by Müller et al. of the Memorial Sloan-Kettering Cancer Centre in New York. They used microarray analysis to
elucidate which genes are altered by administration of PDE5-I in the CN injury
model and  identified genes that are
altered in the CN injury model and that are restored towards normal levels with
the use of sildenafil. These genes predominantly belonged to apoptosis,
neurotrophism, and endothelial funtion families [68].


## 4.3. Hyperbaric Oxygen Therapy

Hyperbaric oxygen therapy (HBOT) has been used in the
treatment of a variety of medical conditions, in the urology field especially
for radiation-induced cystitis. Oxygen hypersaturation has been proven to
induce angiogenesis and enhance wound healing based on the excessive supply of
oxygen. Since hypoxia is assumed to be the capital factor in inducing penile
structural changes and erectile dysfunction following cavernous nerve injury,
Müller et al. [69] investigated the effects of hyperbaric oxygen therapy,
inducing hyperoxygenation in this setting. They used a bilateral cavernous
nerve crush-injury Sprague-Dawley rat model which was exposed to ten sessions
of hyperbaric oxygen at three atmospheres for the duration 90 minutes. They measured ICP after
CN stimulation under controlled conditions with continuous systemic blood
pressure measurement, which according to the authors provides an accurate
measurement of erectile response. They found that rats in the HBOT group had
significantly higher mean ICP/MAP ratios compared with the untreated group.
They demonstrated preservation of smooth muscle/collagen ratio with HBOT using Masson’s Trichrome coloring of penile
cross-sections. This study was the first to show a protective effect of HBOT on
erectile function, and literature data is therefore limited [69]. Theoretically, induction of angiogenesis
by HBOT might potentially accelerate tumor growth in cancer patients and
could therefore be unfavourable in patients treated for pelvic malignancies.
This issue was however studied in a mouse model that was developed to
investigate the effect of HBOT on prostate cancer cell growth in patients
treated for radiation-induced hemorrhagic cystitis. The investigators did not
witness increased rates of tumor growth, and they concluded that HBOT in cases
of prostate cancer is not associated with increased oncological risk [70].


## 4.4. Nitric Oxide Donor Therapy

Since neuropraxia causes loss of NOS
containing nerves and hypoxia, resulting in a decrease in available NO and
structural changes, Veliev and associates hypothesised that direct restoration
of NO in the penis could prevent these changes after denervation. This group
investigated the effect of intracorporeal administration of new NO donor
dinitrosyl iron complex in a bilateral CN crush injury group compared to a
sham-operated group and an untreated group CN crush injury group. They used analysis
of mitotic activity of smooth muscle (decreased after CN injury), endothelium,
and fibroblasts (both increased after CN injury) for evaluation of cavernous
fibrosis development and found a stabilisation of fibroblast proliferation and
improvement of smooth muscle mitotic activity in the group treated with
intracavernosal injections of NO donor [71].


## 5. Neuromodulatory Strategies

During radical prostatectomy, even
with the most advanced nerve-sparing dissection techniques, neuropraxia,
stretching, and axotomy of the cavernous nerves are inevitable. Although the peripheral nervous
system is capable of endogenous regeneration, this response is limited and
usually does not allow full recovery of nerve function.  Cavernous nerves have been shown to possess
the capability to regenerate in 6 months. Recently, accumulating evidence
suggests that a return of potency following radical prostatectomy is partially
dependent on regeneration of the cavernous nerves and axonal regeneration in
the remaining neural tissue. Several treatment strategies to improve this
therapeutic neurogenesis have been under investigation in animal models,
including immunophilin ligands, neurotrophins, growth factors and, more
recently, stem cell therapy [45, 72].


### 5.1. Immunophilin Ligands

Immunophilin ligandscyclosporin A and FK506 (Tacrolimus) are short polypeptides which block the
activation of lymphocytes and other immune cells. In cells, FK506 and cyclosporine
A bind to intracellular proteins called immunophilins. The formed complex inhibits
the activity of calcineurin, a cellular signaling protein which is one of the
key enzymes regulating the activity of many proteins in neurons [71].
Immunophilin ligands have been shown to hold neuroprotective and
neuroregenerative potential in the central nervous system as well as in
peripheral nerves. FK506 showed both functional recovery and nerve regeneration
in the sciatic nerve after crush injury in an animal model [73, 74]. This
success brought immunophilin ligands under interest for the protection of
penile innervation following CN injury. Administration of FK506 orally or
intraperitoneally was found to protect erectile function measured by
intracavernous pressure monitoring after electrical stimulation of the CN
following CN injury. Immunohistochemical evaluation of a CN crush model treated
with FK506 showed maintained peripheral unmyelinated axon integrity and CN
architecture in a bilateral nerve crush injury rat model. The crush injury was
used to simulate the effects of nerve-sparing radical prostatectomy, where
neuropraxia occurs more likely than neurotomy [75, 76]. FK506 also induced high
glutathione peroxidase expression, an antioxidant enzyme, suggesting that FK506
promotes erectile recovery through antioxidative mechanisms [77].


### 5.2. Nonimmunosuppressant Lmmunophilin Ligands

There are some concerns about the
application of immunosuppressant (and carcinogenic) qualities of FK506 therapy
in prostate cancer patients, although daily administration of a dose of 2-3 mg/day in a study by Fleischmann et al. in humans with rheumatoid arthritis did
not induce immunosuppression [78]. Therefore, the use of nonimmunosuppressant
immunophilin ligands has recently gained interest of researchers.
Nonimmunosuppressant molecules have recently been developed: GPI1046 by (Guilford Pharmaceuticals
Inc., Md, USA) (now MGI Pharma), FK1706 by (Astellas Pharmaceuticals Inc., Ill, USA).
Valentine et al. studied the effects of GPI1046 administration on CN integrity
and erectile function under various conditions of CN crush injury. They found
that oral or intraperitoneal administration of GPI1046 preserved intracavernous
pressure (ICP) after electrical CN stimulation, compared to vehicle treated
animals. They also evaluated ultrastructural integrity of the distal CN and
found preservation of unmyelinated axons compared to the vehicle-treated group
[79]. GPI1406 was shown not to promote prostate cancer cells in vitro [80]. Bella et al. proved that subcutaneous administration of FK1706 was effective in
enhancing the recovery of erectile function in a concentration-dependent
manner. With a high-dose treatment group showing significantly increased ICP
compared to the vehicle-treated group in response to electrical CN stimulation.
Sham treated animals showed regular axon sizes and shapes with homogenous
GAP-43 and neurofilament staining, whereas injured axons showed irregular
shapes, sizes, and staining patterns. FK1706 treatment restored axon shape and
staining patterns. Injury significantly decreased nicotinamide adenine dinucleotide
phosphate staining and FK1706 treatment showed a trend toward increased
staining [81, 82].


### 5.3. Neurotrophins and Growth Factors

The effect of growth factors on
erectile recovery and nerve regeneration has been extensively studied by Lue’s
group at the Knuppe Laboratory for Molecular Urology in San Fransisco. They
previously reported enhanced NOS-containing nerve fiber regeneration after
injury, using injections of systemic growth hormone (GH) and insulin-like
growth factor-1 (IGF-1). However, some obvious concerns are existing about the
systemic use of growth factors in patients treated for malignancy.


#### 5.3.1. Brain Derived Neurotrophic Factor

Neurotrophins are proteins that
enhance neuronal regeneration and survival of specific neuronal populations through a variety of
signaling pathways [77]. Brain derived neurotrophic factor (BDNF) has been the
focus of intense investigation because of the central role it plays in neuronal
development, maturation, and survival after injury [72]. It is part of the
family of growth factors that includes NGF and neurotrophin-3, -4, and -5. It
has demonstrable retrograde axonal transport, which occurs as molecules that are
taken up by the neural synapses at the corpus cavernosum and travel retrograde
to the cell body in the major pelvic ganglion to induce their neuroregenerative
effects. This characteristic of BDNF makes it a suitable molecule for
intracavernous injection. Lue’s group demonstrated that intracavernous
injection of adeno-associated virus-BDNF prevents the degeneration of neuronal
NOS containing neurons in the major pelvic ganglion and facilitated
regeneration of  NOS containing nerve
fibers in penile tissue, thus enhancing erectile recovery following bilateral CN
injury. They identified the JAK/STAT pathway as primary mechanism for in vitro
BDNF-mediated neurite outgrowth. They also demonstrated a synergistic effect of
BDNF when combined with vascular endothelial growth factor (VEGF) on neural and
erectile recovery [83].


#### 5.3.2. Glial Cell-Line Derived Neurotrophic Factors

Glial cell-line derived neurotrophic
factors (GDNFs) are a novel group of neuroregenerative molecules of which the
most studied are GDNF and neurturin (NRTN). Studies performed by Leitner et al.
and Laurikainen et al. showed retrograde axonal transport of NRTN when injected
in the penile shaft and suggested an effect of NRTN as a target-derived
neuritogenic factor for postganglionic neurons via a system mediated by the
GDNF receptor family, mainly the GDNF-α2 receptor [72, 84, 85]. In vivo, NRTN was
able to preserve erectile function 5 weeks after nerve crush injury when
applied to the site of nerve injury compared to a control group where albumin
was applied locally, with an ICP increase of 55% compared to the control group
[86].


#### 5.3.3. Growth Differentiation Factor 5

Growth differentiation factor-5
(GDF-5) is a recently isolated neurotrophic factor which belongs to the TGF-β
superfamily, which was previously proven to be a neurotrophic and
neuroprotective factor on dopaminergic neurons after intracerebral injection in
vitro and in vivo. Data concerning the peripheral nervous system is limited to
sensory neurons. Fandel et al. [87] investigated the in vivo effects of
implantation of a GDF-5 impregnated sponge in the corpus cavernosum of a rat CN
crush injury model. They found that intracavernosal application of GDF-5
enhanced the recovery of erectile function and preservation of n-NOS containing
nerve fibers, with a low-dose application giving the most promising results
[87].


#### 5.3.4. Erythropoietin

Erythropoietin (Epo) was originally known for the erythropoiesis.
It is has now become evident that Epo is a multifunctional trophic factor. Epo
for example has potent neurotrophic activity on a variety of neural cells and
includes prevention of cell death and induction of neuronal differentiation.
Campana and Myers demonstrated the production of Epo and Epo receptors in cells
of the peripheral nerve system [88]. This group proposed a model in which
Schwann cells are a major target for Epo in injured peripheral nerves, and
stated that Epo-receptor-induced cell signaling and Schwann cell proliferation
may protect injured peripheral nerves and promote regeneration [89]. Allaf et
al. were the first to show expression of Epo receptor in neuronal cell bodies
of the major pelvic ganglion, penile nerves, and endothelial cells in the
penis, and more recently, in the periprostatic neurovascular bundles [90, 91].
They investigated the potential benefits of Epo on erectile recovery following
cavernous nerve injury, and found significantly greater maximal intracavernous
pressure area under the curve normalized to mean arterial pressure in EPO
treated versus saline treated animals. Morphologically, electron microscopy
revealed significant improvement in axonal regeneration in rhEPO treated
animals 14 days following injury compared to controls [90].


### 5.4. Stem Cell Therapy

Although the number is still
limited, some studies on stem cell therapy for erectile dysfunction have
recently been conducted. Human mesenchymal stem cells (B10) and neural crest
stem cells (K10), transplanted via a retroviral vector into the rat corpus
cavernosum, have been shown to posess the ability to differentiate into smooth
muscle cells or endothelial cells [92, 93]. Damage to smooth muscle and
endothelium plays a major role in the development of veno-occlusive dysfunction
and ED following radical prostatectomy. Therefore B10 and K10 cells may be an
ideal cell source for reconstructing endothelial and smooth muscle cells in the
corpus cavernosum in cell therapy for neurogenic ED. Bochinski et al. used
embryonic stem cells that differentiated along the neuronal cell line, which
they injected into the corpus cavernosum of a rat bilateral CN crush injury
model. They measured a markedly increased ICP upon electrical CN stimulation
compared to controls, and a greater degree of neuroregeneration of n-NOS
containing nerve fibers in the stem cell treatment group [94]. Bella et al. were able to demonstrate
in vitro neurite outgrowth from the major pelvic ganglion of the rat induced by
adult adipose tissue-derived stem cells, which were not induced towards a specific neuronal cell
line prior to administration [95].


## 5.1. Immunophilin Ligands

Immunophilin ligandscyclosporin A and FK506 (Tacrolimus) are short polypeptides which block the
activation of lymphocytes and other immune cells. In cells, FK506 and cyclosporine
A bind to intracellular proteins called immunophilins. The formed complex inhibits
the activity of calcineurin, a cellular signaling protein which is one of the
key enzymes regulating the activity of many proteins in neurons [71].
Immunophilin ligands have been shown to hold neuroprotective and
neuroregenerative potential in the central nervous system as well as in
peripheral nerves. FK506 showed both functional recovery and nerve regeneration
in the sciatic nerve after crush injury in an animal model [73, 74]. This
success brought immunophilin ligands under interest for the protection of
penile innervation following CN injury. Administration of FK506 orally or
intraperitoneally was found to protect erectile function measured by
intracavernous pressure monitoring after electrical stimulation of the CN
following CN injury. Immunohistochemical evaluation of a CN crush model treated
with FK506 showed maintained peripheral unmyelinated axon integrity and CN
architecture in a bilateral nerve crush injury rat model. The crush injury was
used to simulate the effects of nerve-sparing radical prostatectomy, where
neuropraxia occurs more likely than neurotomy [75, 76]. FK506 also induced high
glutathione peroxidase expression, an antioxidant enzyme, suggesting that FK506
promotes erectile recovery through antioxidative mechanisms [77].


## 5.2. Nonimmunosuppressant Lmmunophilin Ligands

There are some concerns about the
application of immunosuppressant (and carcinogenic) qualities of FK506 therapy
in prostate cancer patients, although daily administration of a dose of 2-3 mg/day in a study by Fleischmann et al. in humans with rheumatoid arthritis did
not induce immunosuppression [78]. Therefore, the use of nonimmunosuppressant
immunophilin ligands has recently gained interest of researchers.
Nonimmunosuppressant molecules have recently been developed: GPI1046 by (Guilford Pharmaceuticals
Inc., Md, USA) (now MGI Pharma), FK1706 by (Astellas Pharmaceuticals Inc., Ill, USA).
Valentine et al. studied the effects of GPI1046 administration on CN integrity
and erectile function under various conditions of CN crush injury. They found
that oral or intraperitoneal administration of GPI1046 preserved intracavernous
pressure (ICP) after electrical CN stimulation, compared to vehicle treated
animals. They also evaluated ultrastructural integrity of the distal CN and
found preservation of unmyelinated axons compared to the vehicle-treated group
[79]. GPI1406 was shown not to promote prostate cancer cells in vitro [80]. Bella et al. proved that subcutaneous administration of FK1706 was effective in
enhancing the recovery of erectile function in a concentration-dependent
manner. With a high-dose treatment group showing significantly increased ICP
compared to the vehicle-treated group in response to electrical CN stimulation.
Sham treated animals showed regular axon sizes and shapes with homogenous
GAP-43 and neurofilament staining, whereas injured axons showed irregular
shapes, sizes, and staining patterns. FK1706 treatment restored axon shape and
staining patterns. Injury significantly decreased nicotinamide adenine dinucleotide
phosphate staining and FK1706 treatment showed a trend toward increased
staining [81, 82].


## 5.3. Neurotrophins and Growth Factors

The effect of growth factors on
erectile recovery and nerve regeneration has been extensively studied by Lue’s
group at the Knuppe Laboratory for Molecular Urology in San Fransisco. They
previously reported enhanced NOS-containing nerve fiber regeneration after
injury, using injections of systemic growth hormone (GH) and insulin-like
growth factor-1 (IGF-1). However, some obvious concerns are existing about the
systemic use of growth factors in patients treated for malignancy.


### 5.3.1. Brain Derived Neurotrophic Factor

Neurotrophins are proteins that
enhance neuronal regeneration and survival of specific neuronal populations through a variety of
signaling pathways [77]. Brain derived neurotrophic factor (BDNF) has been the
focus of intense investigation because of the central role it plays in neuronal
development, maturation, and survival after injury [72]. It is part of the
family of growth factors that includes NGF and neurotrophin-3, -4, and -5. It
has demonstrable retrograde axonal transport, which occurs as molecules that are
taken up by the neural synapses at the corpus cavernosum and travel retrograde
to the cell body in the major pelvic ganglion to induce their neuroregenerative
effects. This characteristic of BDNF makes it a suitable molecule for
intracavernous injection. Lue’s group demonstrated that intracavernous
injection of adeno-associated virus-BDNF prevents the degeneration of neuronal
NOS containing neurons in the major pelvic ganglion and facilitated
regeneration of  NOS containing nerve
fibers in penile tissue, thus enhancing erectile recovery following bilateral CN
injury. They identified the JAK/STAT pathway as primary mechanism for in vitro
BDNF-mediated neurite outgrowth. They also demonstrated a synergistic effect of
BDNF when combined with vascular endothelial growth factor (VEGF) on neural and
erectile recovery [83].


### 5.3.2. Glial Cell-Line Derived Neurotrophic Factors

Glial cell-line derived neurotrophic
factors (GDNFs) are a novel group of neuroregenerative molecules of which the
most studied are GDNF and neurturin (NRTN). Studies performed by Leitner et al.
and Laurikainen et al. showed retrograde axonal transport of NRTN when injected
in the penile shaft and suggested an effect of NRTN as a target-derived
neuritogenic factor for postganglionic neurons via a system mediated by the
GDNF receptor family, mainly the GDNF-α2 receptor [72, 84, 85]. In vivo, NRTN was
able to preserve erectile function 5 weeks after nerve crush injury when
applied to the site of nerve injury compared to a control group where albumin
was applied locally, with an ICP increase of 55% compared to the control group
[86].


### 5.3.3. Growth Differentiation Factor 5

Growth differentiation factor-5
(GDF-5) is a recently isolated neurotrophic factor which belongs to the TGF-β
superfamily, which was previously proven to be a neurotrophic and
neuroprotective factor on dopaminergic neurons after intracerebral injection in
vitro and in vivo. Data concerning the peripheral nervous system is limited to
sensory neurons. Fandel et al. [87] investigated the in vivo effects of
implantation of a GDF-5 impregnated sponge in the corpus cavernosum of a rat CN
crush injury model. They found that intracavernosal application of GDF-5
enhanced the recovery of erectile function and preservation of n-NOS containing
nerve fibers, with a low-dose application giving the most promising results
[87].


### 5.3.4. Erythropoietin

Erythropoietin (Epo) was originally known for the erythropoiesis.
It is has now become evident that Epo is a multifunctional trophic factor. Epo
for example has potent neurotrophic activity on a variety of neural cells and
includes prevention of cell death and induction of neuronal differentiation.
Campana and Myers demonstrated the production of Epo and Epo receptors in cells
of the peripheral nerve system [88]. This group proposed a model in which
Schwann cells are a major target for Epo in injured peripheral nerves, and
stated that Epo-receptor-induced cell signaling and Schwann cell proliferation
may protect injured peripheral nerves and promote regeneration [89]. Allaf et
al. were the first to show expression of Epo receptor in neuronal cell bodies
of the major pelvic ganglion, penile nerves, and endothelial cells in the
penis, and more recently, in the periprostatic neurovascular bundles [90, 91].
They investigated the potential benefits of Epo on erectile recovery following
cavernous nerve injury, and found significantly greater maximal intracavernous
pressure area under the curve normalized to mean arterial pressure in EPO
treated versus saline treated animals. Morphologically, electron microscopy
revealed significant improvement in axonal regeneration in rhEPO treated
animals 14 days following injury compared to controls [90].


## 5.3.1. Brain Derived Neurotrophic Factor

Neurotrophins are proteins that
enhance neuronal regeneration and survival of specific neuronal populations through a variety of
signaling pathways [77]. Brain derived neurotrophic factor (BDNF) has been the
focus of intense investigation because of the central role it plays in neuronal
development, maturation, and survival after injury [72]. It is part of the
family of growth factors that includes NGF and neurotrophin-3, -4, and -5. It
has demonstrable retrograde axonal transport, which occurs as molecules that are
taken up by the neural synapses at the corpus cavernosum and travel retrograde
to the cell body in the major pelvic ganglion to induce their neuroregenerative
effects. This characteristic of BDNF makes it a suitable molecule for
intracavernous injection. Lue’s group demonstrated that intracavernous
injection of adeno-associated virus-BDNF prevents the degeneration of neuronal
NOS containing neurons in the major pelvic ganglion and facilitated
regeneration of  NOS containing nerve
fibers in penile tissue, thus enhancing erectile recovery following bilateral CN
injury. They identified the JAK/STAT pathway as primary mechanism for in vitro
BDNF-mediated neurite outgrowth. They also demonstrated a synergistic effect of
BDNF when combined with vascular endothelial growth factor (VEGF) on neural and
erectile recovery [83].


## 5.3.2. Glial Cell-Line Derived Neurotrophic Factors

Glial cell-line derived neurotrophic
factors (GDNFs) are a novel group of neuroregenerative molecules of which the
most studied are GDNF and neurturin (NRTN). Studies performed by Leitner et al.
and Laurikainen et al. showed retrograde axonal transport of NRTN when injected
in the penile shaft and suggested an effect of NRTN as a target-derived
neuritogenic factor for postganglionic neurons via a system mediated by the
GDNF receptor family, mainly the GDNF-α2 receptor [72, 84, 85]. In vivo, NRTN was
able to preserve erectile function 5 weeks after nerve crush injury when
applied to the site of nerve injury compared to a control group where albumin
was applied locally, with an ICP increase of 55% compared to the control group
[86].


## 5.3.3. Growth Differentiation Factor 5

Growth differentiation factor-5
(GDF-5) is a recently isolated neurotrophic factor which belongs to the TGF-β
superfamily, which was previously proven to be a neurotrophic and
neuroprotective factor on dopaminergic neurons after intracerebral injection in
vitro and in vivo. Data concerning the peripheral nervous system is limited to
sensory neurons. Fandel et al. [87] investigated the in vivo effects of
implantation of a GDF-5 impregnated sponge in the corpus cavernosum of a rat CN
crush injury model. They found that intracavernosal application of GDF-5
enhanced the recovery of erectile function and preservation of n-NOS containing
nerve fibers, with a low-dose application giving the most promising results
[87].


## 5.3.4. Erythropoietin

Erythropoietin (Epo) was originally known for the erythropoiesis.
It is has now become evident that Epo is a multifunctional trophic factor. Epo
for example has potent neurotrophic activity on a variety of neural cells and
includes prevention of cell death and induction of neuronal differentiation.
Campana and Myers demonstrated the production of Epo and Epo receptors in cells
of the peripheral nerve system [88]. This group proposed a model in which
Schwann cells are a major target for Epo in injured peripheral nerves, and
stated that Epo-receptor-induced cell signaling and Schwann cell proliferation
may protect injured peripheral nerves and promote regeneration [89]. Allaf et
al. were the first to show expression of Epo receptor in neuronal cell bodies
of the major pelvic ganglion, penile nerves, and endothelial cells in the
penis, and more recently, in the periprostatic neurovascular bundles [90, 91].
They investigated the potential benefits of Epo on erectile recovery following
cavernous nerve injury, and found significantly greater maximal intracavernous
pressure area under the curve normalized to mean arterial pressure in EPO
treated versus saline treated animals. Morphologically, electron microscopy
revealed significant improvement in axonal regeneration in rhEPO treated
animals 14 days following injury compared to controls [90].


## 5.4. Stem Cell Therapy

Although the number is still
limited, some studies on stem cell therapy for erectile dysfunction have
recently been conducted. Human mesenchymal stem cells (B10) and neural crest
stem cells (K10), transplanted via a retroviral vector into the rat corpus
cavernosum, have been shown to posess the ability to differentiate into smooth
muscle cells or endothelial cells [92, 93]. Damage to smooth muscle and
endothelium plays a major role in the development of veno-occlusive dysfunction
and ED following radical prostatectomy. Therefore B10 and K10 cells may be an
ideal cell source for reconstructing endothelial and smooth muscle cells in the
corpus cavernosum in cell therapy for neurogenic ED. Bochinski et al. used
embryonic stem cells that differentiated along the neuronal cell line, which
they injected into the corpus cavernosum of a rat bilateral CN crush injury
model. They measured a markedly increased ICP upon electrical CN stimulation
compared to controls, and a greater degree of neuroregeneration of n-NOS
containing nerve fibers in the stem cell treatment group [94]. Bella et al. were able to demonstrate
in vitro neurite outgrowth from the major pelvic ganglion of the rat induced by
adult adipose tissue-derived stem cells, which were not induced towards a specific neuronal cell
line prior to administration [95].


## 6. Conclusion

Although nerve-sparing techniques
are rapidly emerging, ED following radical prostatectomy remains a major
problem for the prostate cancer patient and his urologist. Disease-free
survival remains the primary goal of radical prostatectomy, although since
patients are increasingly younger at the time of diagnosis and treatment, more
attention is being paid to quality of life issues. The underlying mechanisms
leading to ED have been elucidated by various research institutes. These
insights have led to the use of intracavernosal vasoactive substances and more
recently, oral PDE5-I on a daily basis in patients that develop ED. Currently,
no prophylactic treatment is administered at the time of CN injury. The
benefits of these therapeutic modalities have been demonstrated in preclinical
as well as in clinical studies. More recently, however, the focus of research
has been on the effects of neuromodulatory molecules and stem cell therapy.
Until now, this new class of agents has only been shown to be effective in
reclinical experiments, although FDA approval has been given for the use of
nonimmunosupressant immunophilin ligands. Stem cell therapy is however still
experimental. The increasing insights in mechanisms behind ED following radical
prostatectomy and the identification of novel substances that promote CN
recovery may lead to a new spectrum of treatment modalities for ED following
radical prostatectomy.



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*Source: 594868-2008-06-25.xml*