Functional Connectome of the Fetal Brain.

Large-scale functional connectome formation and reorganization is apparent in the second trimester of pregnancy, making it a crucial and vulnerable time window in connectome development. Here we identified which architectural principles of functional connectome organization are initiated before birth, and contrast those with topological characteristics observed in the mature adult brain. A sample of 105 pregnant women participated in human fetal resting-state fMRI studies (fetal gestational age between 20 and 40 weeks). Connectome analysis was used to analyze weighted network characteristics of fetal macroscale brain wiring. We identified efficient network attributes, common functional modules, and high overlap between the fetal and adult brain network. Our results indicate that key features of the functional connectome are present in the second and third trimesters of pregnancy. Understanding the organizational principles of fetal connectome organization may bring opportunities to develop markers for early detection of alterations of brain function.SIGNIFICANCE STATEMENT The fetal to neonatal period is well known as a critical stage in brain development. Rapid neurodevelopmental processes establish key functional neural circuits of the human brain. Prenatal risk factors may interfere with early trajectories of connectome formation and thereby shape future health outcomes. Recent advances in MRI have made it possible to examine fetal brain functional connectivity. In this study, we evaluate the network topography of normative functional network development during connectome genesis in utero Understanding the developmental trajectory of brain connectivity provides a basis for understanding how the prenatal period shapes future brain function and disease dysfunction.

Fetal growth restriction: current knowledge.

BACKGROUND: Fetal growth restriction (FGR) is a condition that affects 5-10% of pregnancies and is the second most common cause of perinatal mortality. This review presents the most recent knowledge on FGR and focuses on the etiology, classification, prediction, diagnosis, and management of the condition, as well as on its neurological complications. METHODS: The Pubmed, SCOPUS, and Embase databases were searched using the term "fetal growth restriction". RESULTS: Fetal growth restriction (FGR) may be classified as early or late depending on the time of diagnosis. Early FGR (<32 weeks) is associated with substantial alterations in placental implantation with elevated hypoxia, which requires cardiovascular adaptation. Perinatal morbidity and mortality rates are high. Late FGR (≥32 weeks) presents with slight deficiencies in placentation, which leads to mild hypoxia and requires little cardiovascular adaptation. Perinatal morbidity and mortality rates are lower. The diagnosis of FGR may be clinical; however, an arterial and venous Doppler ultrasound examination is essential for diagnosis and follow-up. There are currently no treatments to control FGR; the time at which pregnancy is interrupted is of vital importance for protecting both the mother and fetus. CONCLUSION: Early diagnosis of FGR is very important, because it enables the identification of the etiology of the condition and adequate monitoring of the fetal status, thereby minimizing risks of premature birth and intrauterine hypoxia.

The Relationship of the Temporal Branch of the Facial Nerve to the Fascial Planes of Temporal Region in Human Fetuses.

The aim of this study was to obtain information by assessing the relationship between temporal region fascial structures, fat pads, and temporal branches of facial nerve in human fetuses to use the knowledge on treatment of early childhood period surgeries. This anatomic dissection study was conducted on 40 hemifaces with no visible external abnormalities on their faces. Fascial layers and related fat pads of temporal region were dissected layer by layer beginning from superficial to deep. The relations of temporal branches of facial nerve and temporoparietal fascia and the structures of these fascial layers were evaluated. Temporoparietal fascia showed continuity below zygomatic arc with superficial musculoaponeurotic system. Temporal branches of facial nerve showed a multiple branching. Parotid-masseteric fascia became very thin on the superficial of zygomatic arch and ran with superficial layer of temporal fascia above without attaching to periosteum. Temporal branches of facial nerve entered between multilayered layers of temporoparietal fascia. Temporoparietal fascia became thicker on anterior and middle parts because of the localization of superficial temporal fat pad. Temporal fascia was a 2-layered thick and fibrous tissue enveloping intermediate fat pad. Deep layer connected to periosteum of zygomatic arch and superficial layer continued passing superficial of zygomatic arch and connected to the parotid-masseteric fascia. Deep temporal fat pad was found on the deep to deep layer of temporal fascia and surface of temporal muscle. The findings of this study may contribute to the knowledge of the topographical localization of temporal branches of facial nerve with temporal region fascial structures and fat pads in fetuses.

Identification of the origin of adrenergic and cholinergic nerve fibers within the superior hypogastric plexus of the human fetus.

Nerve fibers contributing to the superior hypogastric plexus (SHP) and the hypogastric nerves (HN) are currently considered to comprise an adrenergic part of the autonomic nervous system located between vertebrae (T1 and L2), with cholinergic aspects originating from the second to fourth sacral spinal segments (S2, S3 and S4). The aim of this study was to identify the origin and the nature of the nerve fibers within the SHP and the HN, especially the cholinergic fibers, using computer-assisted anatomic dissection (CAAD). Serial histological sections were performed at the level of the lumbar spine and pelvis in five human fetuses between 14 and 30 weeks of gestation. Sections were treated with histological staining [hematoxylin-eosin (HE) and Masson's trichrome (TriM)] and with immunohistochemical methods to detect nerve fibers (anti-S100), adrenergic fibers (anti-TH), cholinergic fibers (anti-VAChT) and nitrergic fibers (anti-nNOS). The sections were then digitalized using a high-resolution scanner and the 3D images were reconstructed using winsurf software. These experiments revealed the coexistence of adrenergic and cholinergic fibers within the SHP and the HNs. One-third of these cholinergic fibers were nitrergic fibers [anti-VACHT (+)/anti-NOS (+)] and potentially pro-erectile, while the others were non-nitrergic [anti-VACHT (+)/anti-NOS (-)]. We found these cholinergic fibers arose from the lumbar nerve roots. This study described the nature of the SHP nerve fibers which gives a better understanding of the urinary and sexual dysfunctions after surgical injuries.

Internal anal sphincter parasympathetic-nitrergic and sympathetic-adrenergic innervation: a 3-dimensional morphological and functional analysis.

BACKGROUND: Little detailed information is available concerning morphological and functional autonomic nerve supply to the internal anal sphincter. However, denervation of the sphincter potentially affects anal function after rectal surgery for cancer. OBJECTIVE: The aim of this study was to identify the location and type (nitrergic, adrenergic, and cholinergic) of nerve fibers in the internal anal sphincter and to provide a 3-dimensional representation of their structural relationship in the human fetus. MATERIALS AND METHODS: serial transverse sections were obtained from 14 human fetuses (7 male, 7 female, 15-31 weeks of gestation) and were studied histologically and immunohistochemically; digitized serial sections were used to construct a 3-dimensional representation of the pelvis. MAIN OUTCOMES MEASURES: The location and type of internal anal sphincter nerves were assessed qualitatively. RESULTS: Posteroinferior fibers originating from the inferior hypogastric plexus posteroinferior angle projected to the posterolateral and posterior rectal wall and internal anal sphincter, forming the inferior rectal plexus. The inferior rectal plexus contained vesicular acetylcholine transporter-positive (cholinergic), tyrosine hydroxylase-positive (adrenergic/sympathetic), and neural nitric oxide synthase-positive (nitrergic) fibers. The intrasphincteric vesicular acetylcholine transporter-positive fibers included both neural nitric oxide synthase-negative fibers and neural nitric oxide synthase-positive fibers (nitrergic-parasympathetic). LIMITATIONS: The study focused on topographic and functional anatomy, so that quantitative data were not obtained. A small number of fetal specimens were available. CONCLUSIONS: We report the precise location and distribution of the autonomic neural supply to the internal anal sphincter. This description contributes to the understanding of neurogenic postoperative sphincteric dysfunction. Three-dimensional cartography of pelvic-perineal neurotransmitters provides an anatomical and physiological basis for the selection and development of pharmacological agents to be used in the treatment of primary or postoperative continence and evacuation disorders.

Postnatal neuro-development of fetuses with absent end-diastolic flow in the umbilical artery and/or fetal descending aorta.

OBJECTIVE: To determine whether absence of end-diastolic flow in the umbilical artery and/or fetal aorta impacts postnatal neuro-development in preterm-born children. METHODS: The study group, consisting of 43 fetuses with absent end-diastolic flow in the umbilical artery and/or fetal aorta, was compared with a control group, consisting of 30 fetuses, matching for gestational age but with normal doppler-flow results. The children's neuro-developmental status was assessed using the 'Munich functional developmental diagnostics' (MFDD), between the 2nd and 3rd year of life. RESULTS: Gestational age at birth was 33 + 6 weeks in the study group and 34 + 4 weeks in the control group. A brain-sparing effect was observed in 37.3% of fetuses in the study group compared with 10.0% in the control group (p = 0.014). For all seven MFDD domains, the number of children with deficiencies was higher in the study group. For the domains perception, active speech and comprehension this effect was statistically significant (p < 0.05). Overall, 30.2% of children in the study group and 16.7% of the control group had pathologic test results (p < 0.013). CONCLUSION: Pathological doppler-flow in the umbilical artery and/or fetal descending aorta in preterm born children is associated with neuro-developmental deficiencies. Intensive pediatric care is recommended to mitigate these deficiencies during early childhood.

Anatomical survey of terminal branching patterns of superficial branch of radial nerve in fetuses.

BACKGROUND: Regional anesthesia plays a key role in elective as well as emergency orthopedic and other surgeries in children. However, peripheral nerve blocks are quite challenging in children due to lack of precise anatomical knowledge of the course and distribution of these nerves. The purpose of this study was to explore the terminal branches of the superficial branch of the radial nerve in fetuses. The relationship of the superficial branch of the radial nerve with cephalic vein and surrounding tendons was also observed. METHODS: A total of 60 upper extremities (42 males and 18 females) of 30 spontaneously aborted fetuses (32-40 weeks) were dissected to expose the superficial branch of the radial nerve in the distal forearm and hand. RESULTS: Three patterns of distribution based on the total area of the dorsum of the hand innervated were observed. Type 1 (66.7%): superficial branch of radial nerve (SBRN) innervated lateral two-and-a-half digits; Type 2 (23.3%): SBRN innervated lateral three digits; Type 3 (10%): SBRN innervated lateral three-and-a-half digits. The cephalic vein was seen to intersect the nerve more than twice along its course. CONCLUSIONS: Detailed knowledge of the distribution patterns of terminal branches of superficial branch of radial nerve in hand will enhance the success rate of regional blocks or hand surgeries and minimize the postoperative complications due to injury to nerve or vascular structures.

Gunshot wound of the fetus.

We report here a fetus, who was delivered via cesarean section in 32th gestational week from a 37-year-old mother and sustained multiple gunshots. Post-natal evaluation revealed eight entry-exit holes and the baby was transferred to our clinic.Upon admission, peripheral pulses were nonpalpable, appropriate fluid administration and blood transfusion were conducted and further examinations revealed bladder perforation. Bladder was repaired over a suprapubic catheter and bullet holes were primarily sutured. Left foot drop was observed on follow up and the patient was discharged with no further complication. Although the maternal and the fetal morbidity and mortality rates are high in intrauterine gunshot wounds, appropriate management may provide survival as seen in our case.

Enteric neuronal development in canine small intestine - an immunohistochemical study.

The development of the enteric nervous system (ENS) is still a valid and intensely studied issue. However, literature in the field has no data on this topic in the dog. The present investigations were performed in three groups of fetuses from mongrel dogs - from the third, sixth- -seventh, and ninth week of pregnancy - and in 3-5-day-old puppies (3 specimens for each age group). The tissues (the medial parts of the duodenum, jejunum, and ileum with the cecum and a small portion of the adjacent ascending colon) were cut using a cryostat and the sections were processed for single- and double-labeling immunohistochemistry using antisera against acetylated tubulin (AcTub), vesicular acetylcholine transporter (VAChT), nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), galanin (GAL), neuropeptide Y (NPY), substance P (SP), and calcitonin gene-related peptide (CGRP). In the 3-week-old fetuses, some oval cells invading the gut wall were found. From the seventh week of pregnancy onwards, two different enteric ganglia were present: submucosal and myenteric. The estimated number of nerve elements in the 9-week-old fetuses was much higher than that observed in the 6-7-week-old individuals. There was no significant difference in the estimated number of nerve structures between the 9-week-old fetuses and the 3-5-day-old puppies. The colonization pattern and the development of the ENS in the canine small intestine are very similar to those observed in other mam- mals. However, a few exceptions have been confirmed, regarding the time of appearance of the VIP-, GAL-, and CGRP-immunoreactive neurons, and their distribution in different portions of the canine bowel during development.

The first intestinal motility patterns in fetal mice are not mediated by neurons or interstitial cells of Cajal.

In mature animals, neurons and interstitial cells of Cajal (ICC) are essential for organized intestinal motility. We investigated motility patterns, and the roles of neurons and myenteric ICC (ICC-MP), in the duodenum and colon of developing mice in vitro. Spatiotemporal mapping revealed regular contractions that propagated in both directions from embryonic day (E)13.5 in the duodenum and E14.5 in the colon. The propagating contractions, which we termed ripples, were unaffected by tetrodotoxin and were present in the intestine of embryonic Ret null mutant mice, which lack enteric neurons. Neurally mediated motility patterns were first observed in the duodenum at E18.5. To examine the possible role of ICC-MP, three approaches were used. First, intracellular recordings from the circular muscle of the duodenum did not detect slow wave activity at E16.5, but regular slow waves were observed in some preparations of E18.5 duodenum. Second, spatiotemporal mapping revealed ripples in the duodenum of E13.5 and E16.5 W/W(v) embryos, which lack KIT+ ICC-MP and slow waves. Third, KIT-immunoreactive cells with the morphology of ICC-MP were first observed at E18.5. Hence, ripples do not appear to be mediated by ICC-MP and must be myogenic. Ripples in the duodenum and colon were abolished by cobalt chloride (1 mm). The L-type Ca(2+) channel antagonist nicardipine (2.5 microm) abolished ripples in the duodenum and reduced their frequency and size in the colon. Our findings demonstrate that prominent propagating contractions (ripples) are present in the duodenum and colon of fetal mice. Ripples are not mediated by neurons or ICC-MP, but entry of extracellular Ca(2+) through L-type Ca(2+) channels is essential. Thus, during development of the intestine, the first motor patterns to develop are myogenic.

The developmental anatomy of the human superior hypogastric plexus: A morphometrical investigation with clinical and surgical correlations.

The superior hypogastric plexus (SHP) is the part of the autonomic nervous system, which is responsible for the sympathetic innervation of pelvic organs and extrapelvic genitals in humans of both sexes. The SHP also functions as the anatomic pathway for the major part of visceral sensitive fibers originating from pelvic viscera. In this study, the morphology of the SHP was analyzed through anatomical dissections performed both in human adult and fetal cadavers. A computerized morphometrical investigation of the SHP was also performed and the resulting quantitative data statistically assessed. The comparison between fetal and adult SHP revealed that in the male group there was a developmental increase of six times (in height) and of about five times (in width); while in the female group, there was a developmental increase of 3.5 times both in height and width values. In addition, the distance from the superior border of the SHP to the bifurcation of the common iliac arteries presented a developmental increase of about six times in the male group, and about four times in the female group. We propose an original morphological classification with six types, based upon the anatomical arrangement of the nervous fibers in this autonomic plexus.

Frequency, anatomical properties and innervation of axillary arch and its relation to the brachial plexus in human fetuses.

PURPOSE: "Axillary Arch" (AA) is the main musculotendinous variation of the axillary region. The aim of this study was to reveal the frequency, anatomical properties, and innervation of AA and its relation with brachial plexus in human fetuses. METHODS: In this study, 50 human fetuses (male: 20, female: 30), gestation age varied between 16 and 38 weeks (mean ± SD: 23.3 ± 5.3 week), were dissected at the anatomy laboratory of Mersin University, Faculty of Medicine. RESULTS: AA was found in 11 of the 50 fetuses, and in 3 of 11, it was bilateral. Among both sides, 6 of 14 AAs were on the right and 8 were on the left side. Seven of AA's (7% of the specimens) were in muscular and 7 (7%) of them were in musculotendinous structure. With regard to Testut's classification, six (6%) of them were of complete type, six (6%) were incomplete and two (2%) were concordant with both types. Three different types of AA were defined considering the shapes of the arches. According to our classification, seven of the AAs were of type 1, two of them were of type 2 and five of them were of type 3. Additionally, the incidence of the T2 spinal nerve joining the brachial plexus was significantly higher in the specimens with AA (p = 0.001). CONCLUSIONS: Each type of AA described in this study is thought to have individual clinical significance. Being aware of AA and its types can be important to determine a safe approach to the axillary region for surgeons. In addition, our results show the relation between the occurrence of AA and the variations in the formation of the proximal part of brachial plexus.

The role of the neural sympathetic and parasympathetic systems in diurnal and sleep state-related cardiovascular rhythms in the late-gestation ovine fetus.

The efferent mechanisms mediating the well-known diurnal cardiovascular rhythms in the late-gestation fetus are only partially understood. In the present study, we evaluated the contribution of the parasympathetic and sympathetic nervous systems (SNS) to these rhythms. Chronically instrumented fetal sheep at a mean (SE) of 122 (1) days gestation (term is 147 days) underwent either chemical sympathectomy with 6-hydroxydopamine the day after surgery (n = 8), vagotomy at surgery (n = 8), or were sham controls (n = 8). Fetal heart rate (HR), fetal HR variability (HRV), mean arterial blood pressure (MAP), carotid blood flow (CaBF), electrocorticogram (ECoG) activity, and nuchal activity were measured continuously for 24 h. Changes between sleep states were determined in a 6-h interval. Control fetal sheep showed consistent diurnal rhythms in fetal HR, HRV, MAP, and CaBF, with maximal activity in the evening, but not in nuchal activity. Sympathectomy was associated with a significant reduction of both fetal HR and HRV, while vagotomy was associated with a fall in fetal HRV (P < 0.05) but no change in HR. Despite this, most animals in the two intervention groups still showed diurnal rhythms for fetal HR, HRV, MAP, and CaBF, although peak HR may have been delayed in the sympathectomy group (mean 02:22 vs. 23:54 h in controls, P = 0.06). There was no effect of either intervention on sleep state cycling, although state-related cardiovascular rhythms were significantly modulated. These data indicate that, neither the SNS nor vagal activity, in isolation at least, is essential for generating cardiovascular diurnal rhythms in the late-gestation fetus.

Innervation of the human cervical and thoracic vertebrae at eight postovulatory weeks.

The nerves to the cervical and thoracic vertebrae were traced in 10 serially sectioned human embryos. It was found that the vertebral bodies receive nerve fibres from the trunks of the spinal nerves, anterior branches and meningeal branches of the spinal nerves, and from the sympathetic trunks. Slender twigs from the trunk of the spinal nerve arise close to the spinal ganglion and terminate in the posterior and lateral surfaces of the vertebrae. Fibres from the anterior branches of the spinal nerves terminate in the lateral and anterior surfaces of the vertebrae. Thin rami from the sympathetic trunk reach the anterior surface of the vertebrae.

New insights into fetal pain.

Fetal pain is difficult to assess, because the main feature needed to spot pain, is the subject's capability of declaring it. Nonetheless, much can be affirmed about this issue. In this review we first report the epochs of the development of human nociceptive pathways; then we review since when they are functioning. We also review the latest data about the new topic of analgesia and prenatal surgery and about the scarce effect on fetal pain sentience of the natural sedatives fetuses produce. It appears that pain is a neuroadaptive phenomenon that emerges in the middle of pregnancy, at about 20-22 weeks of gestation, and becomes more and more evident for bystanders and significant for the fetus, throughout the rest of the pregnancy.

Rapid action of estrogens on intracellular calcium oscillations in primate luteinizing hormone-releasing hormone-1 neurons.

Feedback controls of estrogen in LHRH-1 neurons play a pivotal role in reproductive function. However, the mechanism of estrogen action in LHRH-1 neurons is still unclear. In the present study, the effect of estrogens on intracellular calcium ([Ca(2+)](i)) oscillations in primate LHRH-1 neurons was examined. Application of 17beta-estradiol (E(2), 1 nm) for 10 min increased the frequency of [Ca(2+)](i) oscillations within a few minutes. E(2) also increased the frequency of [Ca(2+)](i) synchronization among LHRH-1 neurons. Similar E(2) effects on the frequency of [Ca(2+)](i) oscillations were observed under the presence of tetrodotoxin, indicating that estrogen appears to cause direct action on LHRH-1 neurons. Moreover, application of a nuclear membrane-impermeable estrogen dendrimer conjugate, not control dendrimer, resulted in a robust increase in the frequencies of [Ca(2+)](i) oscillations and synchronizations, indicating that effects estrogens on [Ca(2+)](i) oscillations and their synchronizations do not require their entry into the cell nucleus. Exposure of cells to E(2) in the presence of the estrogen receptor antagonist ICI 182,780 did not change the E(2)-induced increase in the frequency of [Ca(2+)](i) oscillations or the E(2)-induced increase in the synchronization frequency. Collectively, estrogens induce rapid, direct stimulatory actions through receptors located in the cell membrane/cytoplasm of primate LHRH-1 neurons, and this action of estrogens is mediated by an ICI 182,780-insensitive mechanism yet to be identified.

Ontogeny of human intrahepatic innervation.

Intrahepatic nerves serve important metabolic, sensory and motor functions. Their ontogeny in human liver has not been elucidated. We aimed to characterise the ontogeny of human intrahepatic innervation, to assess its relationship with biliary structures and to examine the distribution and nature of peptidergic nerves during development. We used immunohistochemistry on archival normal human liver tissue from 63 fetuses [8-40 gestational weeks (gw)] and 10 adults with antibodies to pan-neural markers and neuropeptides. Few nerve fibers appeared in portal tracts at 8 gw. Their density increased gradually from 12 gw and reached adult levels at 32-33 gw. Rare intra-acinar nerves, restricted to periportal areas, appeared at 40 gw. Galanin-, somatostatin- and calcitonin-gene-related peptide-positive nerve fibers were noted only in portal tracts from 22, 26 and 32 gw, respectively. In human adult liver, dense portal and intra-acinar neural supply was observed. Human fetal liver contains a neural network distributed mainly in portal tracts with a density that increases progressively towards term. Intra-acinar innervation appears at term, suggesting that is not required for normal liver function during development, while peptidergic nerves are important for intrauterine liver functions. Developmentally regulated expression of galanin and somatostatin may play a role in liver morphogenesis.

Enteric nervous system development in cavitary viscera allocated to the celiac plexus.

Enteric nervous system (ENS) is a network made of neuronal cells and nervous fibers. There are two plexuses: myenteric of Auerbach and sub mucous of Meissner and Henle. Many substances are involved in neurotransmission at ENS level. ENS assures all gastrointestinal system functions: digestion, absorption, etc. Our study is made on 23 human fetal specimens at different ages of evolution with crown-rump lengths from 9 to 28 cm, and three new born human specimens. We used the Trichrome Masson stain technique and the argental impregnation Bielschowsky on block technique for microscopic evidence. Our study concerned the cavitary viscera allocated to the celiac plexus, involving all layers of each studied viscera.

Is fetal analgesia necessary during prenatal surgery?

Fetal pain and fetal anesthesia are still matter of debate: some authors hypothesize that several intrauterine endocrine neuroinhibitors (ENIn) anesthetize the fetus, keeping it in a constant state of sleep, and making pharmacological fetal anesthesia useless for fetal surgery, while others argue fetal pain is possible and shoud be prevented with fetal anesthesy. AIM: To retrieve evidences about fetal pain, fetal arousability and about the level of sedation induced by the ENIn, in order to assess the necessity of direct fetal anesthesia during prenatal fetal surgery. METHODS: We performed a careful literature review (1990-2016) on fetal arousability, and on the possibility that ENIn at the average fetal blood levels induce actual anesthesia. We retrieved the papers that fulfilled the research criteria, with particular attention to the second half of pregnancy, the period when most fetal surgery is performed. RESULTS: Fetuses are awake about 10% of the total time in the last gestational weeks, and they can be aroused by external stimuli. ENIn have not an anesthetic effect at normal fetal values, but only when they areartificialy injected at high doses; their blood levels in the last trimester of average pregnancies are not dissimilar either in the fetus or in the mother. CONCLUSIONS: During the second half of the pregnancy, external stimuli can awake the fetuses, although they spend most of the time in sleeping state; the presence of ENIn is absolutely not enough to guarantee an effective anesthesia during surgery. Thus, direct fetal analgesia/anesthesia is mandatory, though further studies on its possible drawbacks are necessary.

Ontogeny of intrinsic innervation in the human thymus and spleen.

The ontogeny of the innervation of human lymphoid organs has not been studied in detail. Our aim was to assess the nature and distribution of parenchymal nerves in human fetal thymus and spleen. We used the peroxidase immunohistochemical technique with antibodies specific to neuron-specific enolase (NSE), neurofilaments (NF), PGP9.5, S100 protein, and tyrosine hydroxylase (TH) and evaluated our results with image analysis. In human fetal thymus, NSE-, NF-, S100-, PGP9.5-, and TH-positive nerves were identified associated with large blood vessels from 18 gestational weeks (gw) onwards, increasing in density during development. Their branches penetrated the septal areas at 20 gw, reaching the cortex and the corticomedullary junction between 20 and 23 gw. Few nerve fibers were seen in the medulla in close association with Hassall's corpuscles. In human fetal spleen, NSE-, NF-, S100-, PGP9.5-, and TH-positive nerve fibers were localized in the connective tissue surrounding the splenic artery at 18 gw. Perivascular NSE-, NF-, S100-, PGP9.5-, and TH-positive nerve fibers were seen extending into the white pulp, mainly in association with the central artery and its branches, increasing in density during gestation. Scattered NSE-, NF-, S100-, PGP9.5-, and TH-positive nerve fibers and endings were localized in the red pulp from 18 gw onward. The predominant perivascular distribution of most parenchymal nerves implies that thymic and splenic innervation may play an important functional role during intrauterine life.